Refrigerator-temperature stable influenza vaccine compositions

ABSTRACT

Methods and compositions for the optimization and production of refrigerator-temperature stable virus, e.g., influenza, compositions are provided. Formulations and immunogenic compositions comprising refrigerator-temperature stable virus compositions are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/487,343, filed onJun. 18, 2009, now U.S. Pat. No. 8,247,207, which is a continuation ofU.S. Ser. No. 11/242,018, filed on Oct. 4, 2005, now abandoned, whichclaims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 60/616,711, filed on Oct. 6, 2004; and is acontinuation-in-part of and claims the benefit under 35 U.S.C. §120 ofU.S. patent application Ser. No. 10/788,236, filed Feb. 25, 2004, nowU.S. Pat. No. 7,262,045, which claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Application No. 60/450,181, filed Feb. 25,2003. All of the forgoing applications are incorporated by reference intheir entirety.

BACKGROUND OF THE INVENTION

Vaccines against various and evolving strains of influenza are importantnot only from a community health stand point, but also commercially,since each year numerous individuals are infected with different strainsand types of influenza virus. Infants, the elderly, those withoutadequate health care and immunocompromised persons are at special riskof death from such infections. Compounding the problem of influenzainfections is that novel influenza strains evolve readily, therebynecessitating the continuous production of new vaccines.

Numerous vaccines capable of producing a protective immune responsespecific for such different influenza viruses have been produced forover 50 years and include, e.g., whole virus vaccines, split virusvaccines, surface antigen vaccines and live attenuated virus vaccines.However, while appropriate formulations of any of these vaccine typesare capable of producing a systemic immune response, live attenuatedvirus vaccines have the advantage of being also able to stimulate localmucosal immunity in the respiratory tract. A vaccine comprising a liveattenuated virus that is capable of being quickly and economicallyproduced and that is capable of easy storage/transport is thus quitedesirable. Even more desirable would be such a vaccine that would becapable of storage/transport at refrigerator temperatures (e.g.,approximately 2-8° C.).

To date, all influenza vaccines commercially available in the U.S. havebeen propagated in embryonated hen eggs. Although influenza virus growswell in hen eggs, the production of vaccine is dependent on theavailability of such eggs. Because the supply of eggs must be organized,and strains for vaccine production selected months in advance of thenext flu season, the flexibility of this approach can be limited, andoften results in delays and shortages in production and distribution.Therefore, methods to increase stability (e.g., at refrigeratortemperatures) of the produced vaccine, are greatly desirable as they canprevent deterioration of vaccine stock, which would otherwisenecessitate new production, etc.

Systems for producing influenza viruses in cell culture have also beendeveloped in recent years (See, e.g., Furminger. Vaccine Production, inNicholson et al. (eds.) Textbook of Influenza pp. 324-332; Merten et al.(1996) Production of influenza virus in cell cultures for vaccinepreparation, in Cohen & Shafferman (eds.) Novel Strategies in Design andProduction of Vaccines pp. 141-151); therefore, any methods to increasevaccine composition stability (e.g., storage/transport at refrigeratortemperature) in these systems as well are also greatly desirable.

Considerable work in the production of influenza virus for production ofvaccines has been done by the present inventors and co-workers; see,e.g., U.S. patent application No. 60/375,675 filed Apr. 26, 2002,PCT/US03/12728 filed Apr. 25, 2003, U.S. Ser. No. 10/423,828 filed Apr.25, 2003, and PCT/US05/017734 filed May 20, 2005.

The present invention provides vaccine compositions that have stabilityat, for example, refrigerator temperatures (e.g., 4° C.) and methods ofproducing the same. Aspects of the current invention are applicable totraditional hen egg and new cell culture vaccine production methods (andalso combined systems) and comprise numerous other benefits that willbecome apparent upon review of the following.

SUMMARY OF THE INVENTION

The current invention provides liquid vaccine formulations that aresubstantially stable at temperatures ranging from 4° C. to 8° C. Theseand other liquid formulations, which are specific embodiments of theinvention are referred to herein, for example, as “vaccine formulationsof the invention,” “refrigerator stable vaccine formulations,” “liquidformulations of the invention,” “formulations of the invention,”“refrigerator-temperature stable (RTS) formulations of the invention,”or simply “compositions of the invention” or “virus compositions of theinvention.”

The present invention provides liquid vaccine formulations that aresubstantially stable at temperatures ranging from 4° C. to 8° C. In onespecific embodiment of the invention, liquid vaccine formulations of theinvention are substantially stable at temperatures ranging from 2° C. to8° C. or at 4° C. for a period of 3, months, or 4 months, or 5 months,or 6 months, or 9 months, or 12 months, or 18 months, or 24 months, or36 months, or 48 months, in that there is an acceptable loss of potency(e.g., influenza virus potency loss), for example, a potency loss ofbetween 0.5-1.0 logs, or less than 0.5 logs, or less than 1.0 logs ofpotency, at the end of such time.

In one embodiment, refrigerator stable vaccine formulations of theinvention are provided that comprise live influenza viruses. Forinstance, formulations of the invention may comprise one or more of thefollowing: an attenuated influenza virus, a cold-adapted influenzavirus, a temperature-sensitive influenza virus, an attenuatedcold-adapted temperature sensitive influenza virus, an influenza Avirus, and an influenza B virus. In one embodiment, liquid vaccineformulations of the invention comprise two influenza A virus strains andone influenza B virus strains.

Alternatively, formulations of the invention may comprise other liveviruses such as paramyxoviruses (e.g., RSV, measles virus, mumps virus,Sendai, New Castle Disease viruses) and parainfluenza virus.

The present invention further provides immunogenic compositionscomprising formulations of the invention. The present invention furtherprovides vaccines (e.g., influenza vaccines) comprising formulations andimmunogenic compositions of the invention.

The present invention further includes methods of producing such liquidvaccine formulations. For instance, in one specific embodiment, methodsof producing liquid formulations comprising one or more influenzaviruses are provided herein. In one specific embodiment, methods ofproducing a liquid formulation of the invention includes one or more ofthe following steps: 1) introducing a plurality of vectors [one or moreof which incorporates (or encodes) a portion of an influenza virusgenome] into a population of host eggs or into a population of hostcells, which population of host eggs or host cells is capable ofsupporting replication of influenza virus; 2) culturing the populationof host eggs or population of host cells at an appropriate temperature;3) recovering influenza viruses in a viral harvest; 4) addition of astabilizer (e.g., sucrose and glutamate-containing solutions asdescribed herein); 5) clarifying the viral harvest (e.g., by depth ormembrane filtration), thereby producing a clarified viral harvest; 6)subjecting the viral harvest to a centrifugation step (e.g., continuouszonal centrifugation, continuous flow centrifugation); 7) a sterilefiltration step (e.g., use of 0.2, or 0.2-0.5 micron filter (with orwithout heating during filtration); and 8) storage at −60 degrees C.

In another specific embodiment, methods of producing a liquidformulation of the invention includes one or more of the followingsteps: 1) infection of a population of host eggs or into a population ofhost cells with influenza viruses; 2) culturing the population of hosteggs or population of host cells at an appropriate temperature; 3)recovering influenza viruses in a viral harvest; 4) addition of astabilizer; 5) clarifying the viral harvest (e.g., by depth filtrationand/or passing through one or more filters ranging from 0.2-0.8 microns;or 0.8 or 1.5 micron followed by 0.2 micron), thereby producing aclarified viral harvest; 6) subjecting the viral harvest to acentrifugation step (e.g., continuous zonal centrifugation, continuousflow centrifugation); 7) a sterile filtration step (e.g., use of 0.2, or0.2-0.5 micron filter (with or without heating during filtration); and8) storage at −60 degrees C.

In other specific embodiment, methods of producing an influenza viruscomposition of the invention comprises one or more of the followingsteps: 1) infection of a population of host eggs or into a population ofhost cells with influenza viruses; 2) culturing the population of hosteggs or population of host cells at an appropriate temperature; 3)recovering influenza viruses in a viral harvest; 4) clarifying the viralharvest by filtration, thereby producing a clarified viral harvest; 5)subjecting the clarified viral harvest to centrifugation (e.g.,continuous flow centrifugation), thereby producing a further clarifiedviral harvest; 6) addition of stabilizers (e.g., one or more of thefollowing: 6-8% sucrose; 1-2% arginine monohydrochloride; 0.05-0.1%glutamic acid, monosodium monohydrate; and 0.5-2% gelatin hydrolysate);and 6) sterilizing said further clarified viral harvest by filtration.

In other specific embodiment, methods of producing an influenza viruscomposition of the invention comprises all of the following steps: 1)infection of a population of host eggs or into a population of hostcells with influenza viruses; 2) culturing the population of host eggsor population of host cells at an appropriate temperature; 3) recoveringinfluenza viruses in a viral harvest; 4) clarifying the viral harvest byfiltration, thereby producing a clarified viral harvest; 5) subjectingthe clarified viral harvest to centrifugation (e.g., continuous flowcentrifugation), thereby producing a further clarified viral harvest;and 6) sterilizing said further clarified viral harvest by filtration.

In other specific embodiment, methods of producing an influenza viruscomposition of the invention comprises one or more of the followingsteps: 1) infection of a population of host eggs or into a population ofhost cells with influenza viruses; 2) culturing the population of hosteggs or population of host cells at an appropriate temperature; 3)recovering influenza viruses in a viral harvest; 4) clarifying the viralharvest; 5) subjecting the clarified viral harvest to centrifugation(e.g., continuous flow centrifugation), thereby producing a furtherclarified viral harvest; and 6) sterilizing said further clarified viralharvest by filtration.

In other specific embodiment, methods of producing an influenza viruscomposition of the invention comprises one or more of the followingsteps: 1) infection of a population of host eggs or into a population ofhost cells with influenza viruses; 2) culturing the population of hosteggs or population of host cells at an appropriate temperature; 3)recovering influenza viruses in a viral harvest; 4) clarifying the viralharvest; and 5) subjecting the clarified viral harvest to diafiltration.

In other specific embodiment, methods of producing an influenza viruscomposition of the invention comprise one or more of the followingsteps: 1) infection of a population of host eggs or into a population ofhost cells with influenza viruses; 2) culturing the population of hosteggs or population of host cells at an appropriate temperature; 3)recovering influenza viruses in a viral harvest; 4) clarifying the viralharvest; 5) subjecting the clarified viral harvest to diafiltration; and6) addition of stabilizers (e.g., one or more of the following: 6-8%sucrose; 1-2% arginine monohydrochloride; 0.05-0.1% glutamic acid,monosodium monohydrate; and 0.5-2% gelatin hydrolysate).

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures appendix.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Displays a flow chart illustrating a CTM Process Flow.

FIG. 2: Displays a flow chart illustrating a CTM Process Flow.

FIG. 3: Displays a table illustrating Centrifuge Loading and TemperatureStudies.

FIG. 4: Displays a table illustrating a Summary of QC test data.

FIG. 5: Displays a table illustrating average yields for a number ofinfluenza strains.

FIG. 6: Displays a table with a summary of QC test data for monovalentbulk release assay results.

DETAILED DESCRIPTION

The present invention provides liquid vaccine formulations that aresubstantially stable at temperatures ranging from 4° C. and 8° C. In onespecific embodiment of the invention, liquid vaccine formulations of theinvention are substantially stable at temperature ranging from 2-8° C.or 4° C. for a period of at least 1 month, or at least 2 months, or atleast 3 months, or at least 4 months, or at least 5 months, or at least6 months, or at least 9 months, or at least 12 months, or at least 18months, or at least 24 months, or at least 36 months, or at least 48months, in that there is an acceptable loss of potency (e.g., influenzavirus potency loss) at the end of such time, for example, a potency lossof between 0.5-1.0 logs (as measured by, e.g., TCID₅₀ or FluorescentFocus Assay (FFA).

The present invention provides liquid vaccine formulations that aresubstantially stable at temperatures ranging from 4° C. and 8° C. In onespecific embodiment of the invention, liquid vaccine formulations of theinvention are substantially stable at temperature ranging from 2-8° C.or 4° C. for a period of at least 1 month, or at least 2 months, or atleast 3 months, or at least 4 months, or at least 5 months, or at least6 months, or at least 9 months, or at least 12 months, or at least 18months, or at least 24 months, or at least 36 months, or at least 48months, in that there is an acceptable loss of potency (e.g., influenzavirus potency loss) at the end of such time, for example, a potency lossof less than 10%, or less than 20%, or less than 30%, or less than 40%,or less than 50%, or less than 60%, or less than 70%, or less than 80%,or less than 90%.

The present invention further provides immunogenic compositionscomprising formulations of the invention. The present invention furtherprovides vaccines (e.g., influenza vaccines) comprising formulationsand/or immunogenic compositions of the invention.

In one embodiment, liquid vaccine formulations of the invention areprovided that comprise live influenza viruses. For instance,formulations of the invention may comprise one or more of the following:an attenuated influenza virus, a cold-adapted influenza virus, atemperature-sensitive influenza virus, an attenuated cold-adaptedtemperature sensitive influenza virus, an influenza A virus, and aninfluenza B virus. In one embodiment, liquid vaccine formulations of theinvention comprise two influenza A virus strains and one influenza Bvirus strains.

Alternatively, formulations of the invention may comprise other liveviruses such as paramyxoviruses (e.g., RSV, parainfluenza virus, measlesvirus, mumps virus, Sendai, New Castle Disease viruses).

The present invention further includes methods of producing such liquidvaccine formulations. For instance, in one specific embodiment, methodsof producing liquid formulations comprising one or more influenzaviruses are provided herein. In one specific embodiment, methods ofproducing a liquid formulation of the invention includes one or more ofthe following steps: 1) introducing a plurality of vectors [one or moreof which incorporates (or encodes) a portion of an influenza virusgenome] into a population of host eggs or into a population of hostcells, which population of host eggs or host cells is capable ofsupporting replication of influenza virus; 2) culturing the populationof host eggs or population of host cells at an appropriate temperature;3) recovering influenza viruses in a viral harvest; 4) addition of astabilizer (e.g., sucrose and glutamate-containing solutions asdescribed herein); 5) clarifying the viral harvest (e.g., by depth ormembrane filtration), thereby producing a clarified viral harvest; 6)subjecting the viral harvest to a centrifugation step (e.g., continuouszonal centrifugation, continuous flow centrifugation); 7) a sterilefiltration step (e.g., use of 0.2, or 0.2-0.5 micron filter (with orwithout heating during filtration); and 8) storage at −60 degrees C.

In another specific embodiment, methods of producing a liquidformulation of the invention includes one or more of the followingsteps: 1) infection of a population of host eggs or into a population ofhost cells with influenza viruses; 2) culturing the population of hosteggs or population of host cells at an appropriate temperature; 3)recovering influenza viruses in a viral harvest; 4) addition of astabilizer; 5) clarifying the viral harvest (e.g., by depth filtrationand/or passing through one or more filters ranging from 0.2-0.8 microns;or 0.8 or 1.5 micron followed by 0.2 micron), thereby producing aclarified viral harvest; 6) subjecting the viral harvest to acentrifugation step (e.g., continuous zonal centrifugation, continuousflow centrifugation); 7) a sterile filtration step (e.g., use of 0.2, or0.2-0.5 micron filter (with or without heating during filtration); and8) storage at −60 degrees C.

In other specific embodiment, methods of producing an influenza viruscomposition of the invention comprises one or more of the followingsteps: 1) infection of a population of host eggs or into a population ofhost cells with influenza viruses; 2) culturing the population of hosteggs or population of host cells at an appropriate temperature; 3)recovering influenza viruses in a viral harvest; 4) clarifying the viralharvest by filtration, thereby producing a clarified viral harvest; 5)subjecting the clarified viral harvest to centrifugation (e.g.,continuous flow centrifugation), thereby producing a further clarifiedviral harvest; 6) addition of stabilizers (e.g., one or more of thefollowing: 6-8% sucrose; 1-2% arginine monohydrochloride; 0.05-0.1%glutamic acid, monosodium monohydrate; and 0.5-2% gelatin hydrolysate);and 6) sterilizing said further clarified viral harvest by filtration.

In other specific embodiment, methods of producing an influenza viruscomposition of the invention comprises all of the following steps: 1)infection of a population of host eggs or into a population of hostcells with influenza viruses; 2) culturing the population of host eggsor population of host cells at an appropriate temperature; 3) recoveringinfluenza viruses in a viral harvest; 4) clarifying the viral harvest byfiltration, thereby producing a clarified viral harvest; 5) subjectingthe clarified viral harvest to centrifugation (e.g., continuous flowcentrifugation), thereby producing a further clarified viral harvest;and 6) sterilizing said further clarified viral harvest by filtration.

In other specific embodiment, methods of producing an influenza viruscomposition of the invention comprises one or more of the followingsteps: 1) infection of a population of host eggs or into a population ofhost cells with influenza viruses; 2) culturing the population of hosteggs or population of host cells at an appropriate temperature; 3)recovering influenza viruses in a viral harvest; 4) clarifying the viralharvest; 5) subjecting the clarified viral harvest to centrifugation(e.g., continuous flow centrifugation), thereby producing a furtherclarified viral harvest; and 6) sterilizing said further clarified viralharvest by filtration.

In other specific embodiment, methods of producing an influenza viruscomposition of the invention comprises one or more of the followingsteps: 1) infection of a population of host eggs or into a population ofhost cells with influenza viruses; 2) culturing the population of hosteggs or population of host cells at an appropriate temperature; 3)recovering influenza viruses in a viral harvest; 4) clarifying the viralharvest; and 5) subjecting the clarified viral harvest to diafiltration.

In other specific embodiment, methods of producing an influenza viruscomposition of the invention comprise one or more of the followingsteps: 1) infection of a population of host eggs or into a population ofhost cells with influenza viruses; 2) culturing the population of hosteggs or population of host cells at an appropriate temperature; 3)recovering influenza viruses in a viral harvest; 4) clarifying the viralharvest; 5) subjecting the clarified viral harvest to diafiltration; and6) addition of stabilizers (e.g., one or more of the following: 6-8%sucrose; 1-2% arginine monohydrochloride; 0.05-0.1% glutamic acid,monosodium monohydrate; and 0.5-2% gelatin hydrolysate).

In one embodiment, methods of producing a liquid formulation of theinvention may include the step of freezing such formulations. Thefreezing step may be done, for example, prior to final stability testingand distribution and/or prior to storage at refrigerator temperatures(e.g., 4-8 degrees Celsius). Freezing the vaccine formulations prior tostorage under refrigerator temperatures may increase stability of thevaccine formulations of the invention by at least 10%, or at least 20%,or at least 30%, or at least 40%, or at least 50%, or at least 80%.

The invention further provides methods of producing one or moreinfluenza virus compositions by filtering an influenza virus harvest,whereby the virus harvest is heated during the filtering. Included inthese specific embodiments, the filtering comprises passage of thecomposition through a microfilter of a pore size ranging from 0.2micrometers to about 0.45 micrometers. Furthermore, in variousembodiments, the temperature of heating in such embodiments optionallycomprises from about 28° C. to about 40° C. or more, while in someembodiments, the temperature comprises 31° C. or from about 30° C. toabout 32° C. The heating in such embodiments optionally occurs before orduring or before and during the filtration and optionally comprises fromabout 50 minutes to about 100 minutes, from about 60 minutes to about 90minutes, or about 60 minutes. The invention also provides an influenzavirus composition produced by such methods (including wherein thecomposition is a vaccine composition).

Stabilizers and Buffers

Stabilizers of the invention include, for example, one or more of thefollowing: arginine (e.g., 0.5-1%, 1-2%; 1%; 1.2%; 1.5%, 0.75-2%);poloxamer; sucrose (e.g., 2-8%; 2%; 6-8%; 3%; 4%; 5%; 6%; 7%, or 8%);hydrolyzed gelatin (e.g., 1%; 0.5-2%; 1.5%; 0.5%; 0.75%); and glutamate(e.g., 0.05-0.1%, 0.02-0.15%, 0.03%, 0.04%, 0.06%, 0.02-0.3%, or 0.094%)

Buffers of the invention include, for example, one or more of thefollowing: phosphate buffer (mono or dibasic or both) (e.g., 10-200 mM,pH 7-7.5; 100 mM, pH 7.2; 100 mM, pH 7-7.3); and histidine buffers(e.g., 25-50 mM histidine, pH 7-7.5, 50-100 mM Histidine, pH 7-7.5).

Process Yield

In one embodiment, methods of producing a liquid formulation of theinvention results in an actual or average process yield (from ViralHarvest (VH) to final formulation) of less than 10%, of less than 16%,less than 20%, less than 30%, less than 40%, less than 50%, less than60%, less than 70%, less than 80%, less than 90%, or less than 93%, orless than 95%.

It will be appreciated by those skilled in the art that the varioussteps of the methods described herein are not required to be performedor required to exist in the same production series. Thus, while in somepreferred embodiments, all steps and/or compositions herein areperformed or exist, in other embodiments, one or more steps areoptionally, e.g., omitted, changed (in scope, order, placement, etc.) orthe like.

It will also be appreciated by those skilled in the art that typicalembodiments comprise steps/methods/compositions that are known in theart, e.g., candling of virus containing eggs, inoculation of eggs withviruses, etc. Therefore, those skilled in the art are easily able todetermine appropriate conditions, sub-steps, step details, etc., forsuch known steps to produce the appropriate viruses, virus solutions,compositions, etc. essentially stable at 2-8° C. The individual stepsare described in greater detail below.

Further, the present invention provides methods of using such liquidvaccine formulations. For example, vaccine formulations may beadministered to a human in order to prevent or reduce the effects of aviral infection, e.g., influenza infection. In one embodiment,formulations of the invention are administered as an immunogeniccomposition to prevent or reduce the effects of an influenza virusinfection.

Refrigerator-Stable CAIV Formulations

Prior work by the inventor and co-workers has resulted in thedevelopment of a trivalent, live, cold-adapted influenza vaccine(CAIV-T, FluMist®, which is referenced throughout as FluMist, but shouldbe assumed to be FluMist®) administered by nasal spray. The currentinvention involves the development of a formulation of CAIV-T, which hasimproved stability profile at refrigerated temperatures. Methods ofproducing such improved formulations are provided herein and may includeone or more of the following steps: a sterile filtration step to reducecontamination risk, ultracentrifugation (e.g., rate zonalcentrifugation), and diafiltration. In addition, included herein are anumber of methods of producing liquid FluMist and otherrefrigerator-temperature stable (RTS) formulations of the invention.

The development of CAIV strains was assisted by Dr. John Maassab of theUniversity of Michigan in the 1960s who serially passaged influenza Aand B strains in PCK cells at decreasing temperatures until theresulting strains reproducibly showed the phenotypic properties ofcold-adaptation (virus grows well at reduced temperatures compared towild type virus), temperature sensitivity (virus does not grow well inelevated temperatures in vitro), and attenuation (virus replication isrestricted in ferrets). Through development by, e.g., the inventor andcoworkers, these properties were used as the basis for development of anannual trivalent vaccine reflecting the CDC-designated vaccines strainsfor a particular year, through the process of 6:2 genetic reassortment.For example, a 6:2 CAIV strain is produced by in vitro co-infection ofthe relevant A or B strain Master Donor Virus (MDV) with the circulatingflu strain of interest, and antibody-mediated selection of the properreassortant. The target 6:2 reassortant contains HA and NA genes fromthe circulating strain, and the remaining genes from the cold adaptedmaster donor virus (MDV). The reassortant retains the cold adaptedphenotypic properties described above. Further development of CAIV hasbeen conducted by the inventors and coworkers. FluMist has demonstrateda safe profile and shown efficacy against viral challenge and isapproved for commercial pharmaceutical use in many situations.

The original formulation of FluMist contained virus harvest (VH)produced by infecting specific pathogen-free chicken eggs withManufacturer's Working Virus Seed (MWVS) of the selected strain,followed by incubation for two to three days, and harvesting infectedallantoic fluid. VH was stabilized by the addition at 1/10^(th) volumeof a 10× sucrose phosphate glutamate (SPG) solution. Trivalent FluMistwas produced by combining VH from each of the three strains in thevaccines for a given year with stabilized normal allantoic fluid (NAF)to a target concentration of 7.3 log₁₀ TCID₅₀/mL of each strain. Theresulting blend was then filled into sprayers fitted with a spray tipallowing intranasal delivery of FluMist vaccine. This product format wasused as “frozen FluMist”, which was stored in a frozen form. It will beappreciated that the MWVS virus could also optionally be manufacturedby, e.g., plasmid reassortment. See, e.g., U.S. patent application No.60/375,675 filed Apr. 26, 2002, PCT/US03/12728 filed Apr. 25, 2003, U.S.Ser. No. 10/423,828 filed Apr. 25, 2003, PCT/US05/017734 filed May 20,2005, and US20050186563.

While frozen FluMist can serve as a marked vaccine when frozen aftermanufacture and held frozen until time of use, a form of FluMist that isstable for transport/storage at refrigerator temperatures is quitedesirable. Such “refrigerator-temperature stable” (or RTS) forms arecharacterized by one or more (but not necessarily all in eachembodiment) of the following: retention of stability when distributed asa refrigerated liquid; are passed through sterilizing filters (e.g., 0.2micron) to provide assurance of a sterile product; have reduced contentof egg protein (e.g., substantially free of NAF); have eliminated theneed for manufacture of NAF diluent; have a reduced volume of a dose;and comprises either or both arginine and gelatin as excipients (e.g.,as stabilizers). In certain aspects herein, formulations having one ormore such characteristics are referred to as “liquid FluMist” or RTS orvarious similar terms, to distinguish from other versions of CAIV-Tvaccine, such as frozen. The current invention presents these and otheraspects.

In producing/testing a liquid RTS virus composition of the invention,numerous development batches were conducted. Development batches rangedfrom 2000 to 20,000 eggs per lot, while GMP batches were approximately10,000 eggs each. It will be appreciated that while various examples andprotocols are given herein for production of MWVS viruses (e.g.,reassortants), the viruses are optionally produced through differentmeans in different embodiments. For example, in certain embodiments, theviruses herein are optionally made through the protocols shown herein,while in other embodiments, the MWVS viruses are optionally madethrough, e.g., plasmid reassortment or “plasmid rescue” technologies.See, e.g., U.S. patent application No. 60/375,675 filed Apr. 26, 2002,PCT/US03/12728 filed Apr. 25, 2003, U.S. Ser. No. 10/423,828 filed Apr.25, 2003, U.S. Ser. No. 10/788,236 filed Feb. 25, 2004, PCT/US05/017734filed May 20, 2005, and US20050186563, which are each incorporated byreference herein. Accordingly, as used herein “infection of a populationof host cells” encompasses host cells infected by virus created by orduring plasmid reassortment.

In various embodiments, the invention comprises virus and vaccinecompositions that are substantially stable, e.g., do not showunacceptable losses in potency, e.g., potency loss of between 0.5-1.0logs, or less than 0.5 logs, or less than 1.0 logs, over selected timeperiods (typically for at least 1 month, for at least 2 months, for atleast 3 months, for at least 4 months, for at least 5 months, for atleast 6 months, for at least 7 months, for at least 8 months, for atleast 9 months, for at least 10 months, for at least 11 months, for atleast 12 months, for at least 13 months, for at least 14 months, for atleast 15 months, for at least 16 months, for at least 17 months, for atleast 18 months, for at least 19 months, for at least 20 months, for atleast 21 months, for at least 22 months, for at least 23 months, or forat least 24 months, or for greater than 24 months, etc.) at desiredtemperatures (e.g., typically 4° C., 5° C., 8° C., from about 2° C. toabout 8° C. or greater than 2° C., or between the ranges of 2° C. to 4°C., or between the ranges of 2° C. to 8° C.).

While a number of aspects of the invention herein are exemplified orillustrated with FluMist, the principles embodied by the invention areapplicable to other virus/vaccine compositions as well and should notnecessarily be limited to particular strains/viruses herein. Thus otherlive attenuated influenza virus and vaccines and compositions are alsowithin the purview of the invention, e.g., ones created through rationalmeans, by human intervention, etc. Also, other viruses of otherinfluenza strains, etc., such as influenza A strains, influenza Bstrains, attenuated and non-attenuated influenza strains, cold adaptedand non-cold adapted influenza strains, temperature sensitive andnon-temperature sensitive influenza strains, etc. are all optionallywithin the embodiments of the current invention. Such other virus andvaccine can be used, e.g., as new vaccine and/or as controls for testingother vaccine either in humans or animals, etc. In addition, other liveviral vaccine compositions particularly those comprising live virusesgrown in chicken cells or eggs (e.g., measles virus) are embodiments ofthe invention. Furthermore, the principles embodied by the invention arealso largely applicable to virus and vaccine compositions comprisinglive viruses grown in mammalian cells. See, e.g., U.S. Pat. Nos.6,244,354; 6,146,873; and 6,656,720.

Bulk Virus Harvest Production

Purification of the cold-adapted influenza virus (or other similarviruses) and the actual formulation of the compositions are features ofRTS, or liquid, virus compositions. Separation of influenza virus fromallantoic fluid had been practiced as a part of commercial processes formanufacture of inactivated vaccines. The method of choice for suchinactivated vaccine has been ultra-centrifugation. Commercial scalecontinuous flow ultracentrifuge became available in 1969 and was quicklyapplied to the preparation of inactivated influenza vaccines. Whilechromatographic purification of live influenza virus would be anattractive alternative, robust large-scale processes that retain viralactivity are not yet available. This is thought to be due to themembrane coat and pleiomorphic nature of the influenza virus particle.

Recovery of live virus (as opposed to inactivated virus) purified byultra-centrifugation was achieved by the inventors and coworkers and isan embodiment of the current invention. Further work demonstrated theability of depth filtration as a commercially viable alternative toswinging-bucket centrifugation prior to ultra-centrifugation, andacceptable recoveries of live virus following filtration through a 0.2micron filter, and again such is an embodiment of the current invention.

In one specific embodiment, the median process yield for the VHclarification step of the methods of the invention is at least 30%, orat least 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or at least 90%.

In another specific embodiment, the median process yield for theultracentrifugation (e.g., zonal centrifugation) step of the methods ofthe invention is at least 30%, or at least 40%, or at least 50%, or atleast 60%, or at least 70%, or at least 80%, or at least 90%.

In another specific embodiment, the median process yield for the peakdilution and sterile filtration step of the methods of the invention isat least 30%, or at least 40%, or at least 50%, or at least 60%, or atleast 70%, or at least 80%, or at least 90%.

The ultra-centrifugation step provides the benefit of concentratingCAIV. This supports the use of a smaller delivered volume (0.1 mL pernostril rather than 0.25 mL per nostril, e.g., as might be used forfrozen FluMist). Such reduced volume is more typical of nasallyadministered products and can increase consumer acceptance and reduceproduct losses due to swallowing or vaccine dripping out of the nose.Infected allantoic fluid harvest titers for CAIV have typically beenbetween 8.3 and 9.5 log₁₀ TCID₅₀/mL, which is more than an order ofmagnitude above the target product concentration for frozen FluMist (7.3log₁₀ TCID₅₀/mL, or 7.0 log₁₀ TCID₅₀ per dose). In the event that a verylow titer strain is included in the annual vaccine recommendation, aliquid or RTS FluMist improves the chances of producing a full strengthtrivalent vaccine despite the increased virus concentration in the finalproduct compared to frozen FluMist. Liquid or RTS FluMist is optionallyformulated to a final concentration of 7.7 log₁₀ TCID₅₀/mL, whichdelivers the same amount of live virus per dose as frozen FluMist.

In one specific embodiment, a low titer influenza vaccine composition isprovided whereby the viral titer is less than 7.3 log₁₀ TCID₅₀/mL, orless than 7.0 log₁₀ TCID₅₀/mL, or less than 6.0 log₁₀ TCID₅₀/mL, or lessthan 5.0 log₁₀ TCID₅₀/mL, or less than 4.0 log₁₀ TCID₅₀/mL, or less than3.0 log₁₀ TCID₅₀/mL, or less than 2.0 log₁₀ TCID₅₀/mL. Such low titerinfluenza vaccine compositions may further comprise a pharmaceuticallyacceptable adjuvant, e.g., E. coli heat-labile toxin (or fragmentsthereof), pertussis toxin, aluminum. Other adjuvants include, but arenot limited to aluminum phosphate, aluminum hydroxide, MPL™.(3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc.,Hamilton, Mont., now Corixa), synthetic lipid A analogs such as 529(Corixa), Stimulon™. QS-21 (Aquila Biopharmaceuticals, Framingham,Mass.), IL-12 (Genetics Institute, Cambridge, Mass.), syntheticpolynucleotides such as oligonucleotides containing a CpG motif (U.S.Pat. No. 6,207,646 (28)), and cholera toxin (either in a wild-type ormutant form, for example, where the glutamic acid at amino acid position29 is replaced by another amino acid, preferably a histidine, inaccordance with published International Patent Application Number WO00/18434).

In one specific embodiment, diafiltration may be used in the preparationof virus compositions of the invention. For instance, diafiltration maybe used to concentrate the virus prior to formulation. Diafiltration maybe used in addition to or instead of ultracentrifugation.

After an initial phase described later herein, cGMP production wasinitiated at the scale of 10,000 eggs per lot. A/Beijig/262/95 (H1N1),A/Sydney/05/97 (H3N2) and B/Ann Arbor/1/94 (B/harbin/7/94-like) purifiedmonovalent CAIV bulks were manufactured to support subsequent clinicaltrials. The B/Ann Arbor strain is hereafter referred to asB/Harbin/7/94-like. For convenience, strain designations are oftenabbreviated (e.g., A/Beijing). The current application describes mostheavily the process steps unique to the liquid FluMist, however, stepsinvolved in both or common to both liquid and frozen FluMist are alsodescribed herein.

Methods for egg management and preparation of fresh virus harvest wereessentially the same in various embodiments of the current invention(with the exception of automated inoculation and harvesting) for frozenFluMist as for liquid FluMist and those of skill in the art will beaware of similar or equivalent steps which are capable of use with thecurrent invention. In the current invention, ultra-centrifugation wasperformed using a Hitachi CP40Y zonal centrifuge and an RP40CT Type Dcontinuous-flow rotor (rotor volume=3.2L). Virus harvest was firstpooled, stabilized with sucrose phosphate glutamate (SPG) to facilitatefiltration, then passed through a 5 micron polypropylene filter. Thefiltrate was then loaded onto a 10% to 60% sucrose gradient, banded forone hour at 40,000 rpm, and eluted into 100-ml fractions.

Fractions containing high hemagglutinin (HA) levels were pooled, dilutedto 0.2M sucrose concentration, and then sterile filtered using apolyvinylidene fluoride (PVDF) 0.2 micron filter. The resulting bulkpurified monovalent CAIV was frozen in 1L bottles below −60 degrees C.and held for further processing.

Formulation and Filing

Initial formulation screening determined that the liquid phase stabilityof purified CAIV at refrigerated temperatures was suitable for an annualvaccine. Hydrolyzed animal gelatin added to the frozen FluMiststabilizer SPG provided the best stability results, with a least stablestrain and lot from the CTM-1 campaign showing a loss of one log over6.9 months. Further formulation development studies showed that thestability of liquid FluMist could be further improved by storing frozenjust after manufacture, and thawing before final distribution. Thestability of the worst-case strain was also improved by addition ofarginine. While animal gelatins can raise concerns with regard totransmissible spongiform encephalopathies (TSE), available formulationslacking gelatin did not achieve the required stability in allembodiments. Thus, porcine gelatin was chosen for the some embodimentsof liquid FluMist formulation due to its stabilizing properties and thefact that there are no reported occurrences of TSE in pigs. The harshchemical processing steps used in collagen hydrolysis are also thoughtto cause inactivation of prion-sized proteins.

Frozen, purified monovalent bulk CAIV was shipped and trivalent vaccinewas produced under cGMP to support clinical testing of liquid FluMist. Atotal of six fills were performed. Blending was performed at small scaleusing a 4-liter glass aspirator flask, and 0.5 mL Becton Dickinson (BD)Hypack SCF (sterile, clean, ready to fill) glass sprayers were filledusing an NOVA automated filler/stopperer and associated equipment. Themanufacture of filled trivalent liquid FluMist is described in moredetail below.

Specific Formulation Embodiments of the Invention

In one embodiment, the vaccine formulations of the invention compriseone or more of the following in the final formulations: sucrose: 6-8%weight/volume (w/v); arginine monohydrochloride 1-2% w/v; glutamic acid,monosodium monohydrate 0.05-0.1% w/v; gelatin hydrolysate, porcine TypeA (or other sources) 0.5-2% w/v; potassium phosphate dibasic 1-2%; andpotassium phosphate monobasic 0.25-1% w/v.

In one specific embodiment, vaccine formulations comprise one or more ofthe following: sucrose: 6.84% weight/volume (w/v); argininemonohydrochloride 1.21% w/v; glutamic acid, monosodium monohydrate 0.094w/v; gelatin hydrolysate, porcine Type A (or other sources) 1% w/v;potassium phosphate dibasic 1.13%; and potassium phosphate monobasic0.48% w/v. In another specific embodiment, vaccine formulations compriseall of the following: sucrose: 6.84% weight/volume (w/v); argininemonohydrochloride 1.21% w/v; glutamic acid, monosodium monohydrate0.094% w/v; gelatin hydrolysate, porcine Type A (or other sources) 1%w/v; potassium phosphate dibasic 1.13%; and potassium phosphatemonobasic b 0.48% w/v.

In another specific embodiment, vaccine formulations comprise all of thefollowing (within 10% variation of one or more component): sucrose:6.84% weight/volume (w/v); arginine monohydrochloride 1.21% w/v;glutamic acid, monosodium monohydrate 0.094% w/v; gelatin hydrolysate,porcine Type A (or other sources) 1% w/v; potassium phosphate dibasic1.13%; and potassium phosphate monobasic 0.48% w/v.

In another specific embodiment, vaccine formulations comprise all of thefollowing (within 10% variation of one or more component): sucrose:6.84% weight/volume (w/v); arginine monohydrochloride 1.21% w/v; gelatinhydrolysate, porcine Type A (or other sources) 1% w/v. In suchembodiments, formulation are in buffer (e.g., a potassium phosphatebuffer (pH 7.0-7.2)).

In another specific embodiment, vaccine formulations comprise all of thefollowing (within 20% variation of one or more component): sucrose:6.84% weight/volume (w/v); arginine monohydrochloride 1.21% w/v;glutamic acid, monosodium monohydrate 0.094% w/v; gelatin hydrolysate,porcine Type A (or other sources) 1% w/v; potassium phosphate dibasic1.13%; and potassium phosphate monobasic 0.48% w/v.

In another specific embodiment, vaccine formulations comprise all of thefollowing (within 30% variation of one or more component): sucrose:6.84% weight/volume (w/v); arginine monohydrochloride 1.21% w/v;glutamic acid, monosodium monohydrate 0.094% w/v; gelatin hydrolysate,porcine Type A (or other sources) 1% w/v; potassium phosphate dibasic1.13%; and potassium phosphate monobasic 0.48% w/v.

In another specific embodiment, vaccine formulations comprise all of thefollowing (within 40% variation of one or more component): sucrose:6.84% weight/volume (w/v); arginine monohydrochloride 1.21% w/v;glutamic acid, monosodium monohydrate 0.094% w/v; gelatin hydrolysate,porcine Type A (or other sources) 1% w/v; potassium phosphate dibasic1.13%; and potassium phosphate monobasic 0.48% w/v.

In another specific embodiment, vaccine formulations comprise all of thefollowing (within 1% variation of one or more component): sucrose: 6.84%weight/volume (w/v); arginine monohydrochloride 1.21% w/v; glutamicacid, monosodium monohydrate 0.094% w/v; gelatin hydrolysate, porcineType A (or other sources) 1% w/v; potassium phosphate dibasic 1.13%; andpotassium phosphate monobasic 0.48% w/v.

In another specific embodiment, vaccine formulations comprise all of thefollowing (within 3% variation of one or more component): sucrose: 6.84%weight/volume (w/v); arginine monohydrochloride 1.21% w/v; glutamicacid, monosodium monohydrate 0.094% w/v; gelatin hydrolysate, porcineType A (or other sources) 1% w/v; potassium phosphate dibasic 1.13%; andpotassium phosphate monobasic 0.48% w/v.

In a specific embodiment, formulations of the invention may contain,e.g., potassium phosphate (e.g., at least 50 mM, or at least 100 mM, orat least 200 mM, or at least 250 mM) as a buffer or alternatively,histidine (e.g., at least 50 mM, or at least 100 mM, or at least 200 mM,or at least 250 mM).

Other Specific Embodiments of the Invention, e.g., Dosing, Potency

In another specific embodiment, methods of administering the vaccineformulations of the invention intranasally are included. For instance,vaccine formulations of the invention may be administered intranasallyin final doses of 0.5 mL/dose; 0.75 mL/does; 0.1 mL/does; 0.15 mL/does;0.2 mL/dose; 0.25 mL/dose; 0.5-0.1 mL/dose; 0.5 mL-2 mL/dose; or 0.5-2.5mL/dose.

In one embodiment, vaccine formulations of the invention compriseapproximately 10⁷ fluorescent focus units (FFU) or 10⁷ TCID₅₀ of each ofthree different reassortant strains of influenza. In another specificembodiment, vaccine formulations of the invention comprise a potency of7.0 (+/−0.5) log10 FFU/dose (or TCID₅₀/dose) for each strain ofinfluenza virus. In another specific embodiment, vaccine formulations ofthe invention comprise a potency of 7.0 (+/−0.5) log10 FFU/dose (orTCID₅₀/dose) for at least one strain of influenza virus. In anotherspecific embodiment, vaccine formulations of the invention have apotency of at least (or equal to) 6.0 log10 FFU/dose, 6.5 log10FFU/dose, 6.7 log10 FFU/dose 6.7 log10 FFU/dose, 6.8 log10 FFU/dose, 6.9log10 FFU/dose, 7.0 log10 FFU/dose, 7.1 log10 FFU/dose, 7.2 log10FFU/dose, 7.3 log10 FFU/dose, 7.4 log10 FFU/dose, 7.5 log10 FFU/dose,7.6 log10 FFU/dose, 7.7 log10 FFU/dose, 7.8 log10 FFU/dose, 7.9 log10FFU/dose, 8.1 log10 FFU/dose, 8.5 log10 FFU/dose or 8.8 log10 FFU/dose.

Potency may be measured by TCID₅₀ instead of FFU in each embodiment ofthe invention. A Fluorescent Focus Assay (FFA) is a direct measure ofthe infectivity in the vaccine while TCID₅₀ is an indirect measure whichmeasures the ability of productive replication in MDCK or other cells.

In another specific embodiment, vaccine formulations of the inventionhave a potency of between 6.5-7.5 log 10 FFU per dose (e.g., per 0.2 mLdose).

In another specific embodiment, vaccine formulations of the inventionhave a potency of at least (or equal to) 7.0 log₁₀ TCID₅₀ per dose, orat least 6-8 TCID₅₀ per dose, or at least 6.4 TCID₅₀ per dose, or atleast 6.6 log₁₀ TCID₅₀ per dose.

In one embodiment, vaccine formulations of the invention have pH of6.7-7.7, or 6.6, or 6.7, or 6.8, or 6.9, or 7.0, or 7.1, or 7.2, or 7.3,or 7.4, or 7.5, or 7.6, or 7.7.

In another specific embodiment, the vaccine formulations of theinvention have no greater than 60 EU/mL of endotoxin, or have no greaterthan 200 EU/mL of endotoxin. For instance, the vaccine formulations ofthe invention have less than or equal to 5 EU/mL of endotoxin. Inanother specific embodiment, the vaccine formulations of the inventionhave less than or equal to 1.0 EU/7.0 log10 FFU.

In another specific embodiment, the vaccine formulations of theinvention are free of one or more (or all) of the following: mycoplasma,retrovirus, avian leucosis virus, and mycobacterium (e.g., M.tuberculosis). Methods of the producing such formulations of theinvention may comprise the step(s) of testing for such impurities.

In another specific embodiment, the vaccine formulations of theinvention may contain influenza viruses having HA and NA genes fromthree different viruses as in FluMist.

In one specific embodiment, formulations of the invention comprise oneor more (or all) of the following per dose (e.g., per 0.2 mL dose):sucrose (13.68 mg), dibasic potassium phosphate (2.26 mg), monobasicpotassium phosphate (0.96 mg), gelatin hydrolysate (2.0 mg), argininehydrochloride (2.42 mg), and monosodium glutamate (0.188 mg).

In one specific embodiment, formulations of the invention comprise oneor more (or all) of the following per dose (e.g., per 0.2 mL dose):sucrose (13.68 mg), potassium phosphate (100 mM), gelatin hydrolysate(2.0 mg), arginine hydrochloride (2.42 mg), and monosodium glutamate(0.188 mg).

In another specific embodiment, formulations of the invention compriseone or more (or all) of the following (plus or minus 10%, or 20% or 30%or 40%) per dose: sucrose (13.68 mg), dibasic potassium phosphate (2.26mg), monobasic potassium phosphate (0.96 mg), gelatin hydrolysate (2.0mg), arginine hydrochloride (2.42 mg), and monosodium glutamate (0.188mg).

In another specific embodiment, formulations of the invention compriseone or more (or all) of the following (plus or minus 10%, or 20% or 30%or 40%) per dose: sucrose (13.68 mg), potassium phosphate (100 mM),gelatin hydrolysate (2.0 mg), arginine hydrochloride (2.42 mg), andmonosodium glutamate (0.188 mg).

In another specific embodiment, formulations of the invention comprisean influenza virus comprising the genetic backbone of one or more of thefollowing influenza viruses: A/Ann Arbor/6/60 (A/AA/6/60) B/AnnArbor/1/66 virus, the FluMist MDV-A (ca A/Ann Arbor/6/60), the FluMistMDV-B (ca B/Ann Arbor/1/66), A/Leningrad/17 donor strain backbone, andPR8.

In another specific embodiment, the vaccine formulations of theinvention comprise an influenza virus comprising an HA and an NApolypeptide sequence (or at least 90% identical or at least 95%identical to such sequences) from one or more of the following:B/Yamanashi; A/New Caledonia; A/Sydney; A/Panama; B/Johannesburg;B/Victoria; B/Hong Kong; A/Shandong/9/93; A/Johannesburg/33/94;A/Wuhan/395/95; A/Sydney/05/97; A/Panama/2007/99; A/Wyoming/03/2003;A/Texas/36/91; A/Shenzhen/227/95; A/Beijing/262/95; A/NewCaledonia/20/99; B/Ann Arbor/1/94; B/Yamanashi/166/98;B_Johannesburg_(—)5_(—)99; BNictoria/504/2000; B/Hong Kong/330/01;B_Brisbane_(—)32_(—)2002; B/Jilin/20/03; an H1N1 influenza A virus, anH3N2 influenza A virus, H9N2 influenza A virus, an H5N1 influenza Avirus; an influenza B virus; and a pandemic influenza strain (eitherdesignated by WHO or not circulating in the human population). See,e.g., US 20050042229.

In another specific embodiment, the vaccine formulations of theinvention are sterile.

Description of Representative Steps in Vaccine Production

For ease in discussion and description, the various steps of vaccinecomposition production in general, can be thought of as comprising orfalling into four broad groups (roughly similar to the presentationoutlined previously above). The first group comprises such aspects asco-infection, reassortment, selection of reassortants, and cloning ofreassortants. The second group comprises such aspects as purificationand expansion of reassortants. The third group comprises furtherexpansion of reassortants in eggs, along with harvesting andpurification of such harvested virus solutions. The fourth groupcomprises stabilization of harvested virus solutions andpotency/sterility assays of the virus solutions. It is to be understood,however, that division of the aspects of the invention into the abovefour general categories is solely for explanatory/organizationalpurposes and no inference of interdependence of steps, etc. should bemade. Again, it will be appreciated that other steps (both similar anddifferent) are optionally used with the methods and compositions of theinvention (e.g., the methods and compositions for RTS vaccinecompositions).

As mentioned above, for ease in discussion and description, the varioussteps of vaccine production can be thought of as comprising four broadgroups. The first group comprises such aspects as co-infection,reassortment, selection of reassortants, and cloning of reassortants.

Group 1

The aspects of vaccine composition production which are broadlyclassified herein as belonging to Group 1, comprise methods andcompositions related to optimization of co-infection of cell culturelines, e.g., with a master donor virus and one or more wild-type virusesin order to produce specifically desired reassorted viruses; selectionof appropriate reassorted viruses; and cloning of the selectedreassorted viruses. Reassortment of influenza virus strains is wellknown to those of skill in the art. Reassortment of both influenza Avirus and influenza B virus has been used both in cell culture and ineggs to produce reassorted virus strains. See, e.g., Tannock et al.,Preparation and characterisation of attenuated cold-adapted influenza Areassortants derived from the A/Leningrad/134/17/57 donor strain,Vaccine (2002) 20:2082-2090. Reassortment of influenza strains has alsobeen shown with plasmid constructs. See, e.g., U.S. patent applicationNo. 60/375,675 filed Apr. 26, 2002, PCT/US03/12728 filed Apr. 25, 2003and U.S. application Ser. No. 10/423,828, filed Apr. 25, 2003,PCT/US05/017734, filed May 20, 2005; and US20050186563.

Reassortment, in brief, generally comprises mixing (e.g., in eggs orcell culture) of gene segments from different viruses. For example, thetypical 8 segments of influenza B virus strains can be mixed between,e.g., a wild-type strain having an epitope of interest and a “donor”strain, e.g., comprising a cold-adapted strain. Reassortment between thetwo virus types can produce, inter alia, viruses comprising thewild-type epitope strain for one segment, and the cold-adapted strainfor the other segments. Unfortunately, to create the desiredreassortants, sometimes large numbers of reassortments need to be done.After being reassorted, the viruses can also be selected (e.g., to findthe desired reassortants). The desired reassortants can then be cloned(e.g., expanded in number).

Traditional optimization, selection, and cloning of desired reassortantsfor influenza B virus, typically occurs by co-infection of virus strainsinto a cell culture (e.g., CEK cells) followed by selection withappropriate antibodies, e.g., against material from one of the parentvirus, (usually done in eggs), and cloning or expanding of virus, etc.which is typically done in cell culture. However, such traditionalreassortment presents drawbacks in that thousands of reassortments areneeded to create the desired segment mix. When such reassortments aredone, it is apparent that truly random reassortments are not the endresult. In other words, pressures that bias the process exist in thesystems. For influenza A strains, however, such processes do not appearto have such bias. For A strains, co-infection of strains (typicallyinto cell culture such as CEK cells) is followed by selection andcloning at the same time, again, typically in cell culture.

Optimization of Reassortment

Various embodiments utilizing the steps in Group 1 can optimize thereassortment process in order to reduce the number of reassortmentsneeded (and thus increase the throughput/stability of the vaccineproduction process), etc. The steps utilizing such optimizationtechniques are typically embodied with reassortment of influenza Bstrains and are typically done in cell culture, e.g., CEK cells. See,e.g., U.S. patent application Ser. No. 10/788,236 and PCT/US04/05697both filed Feb. 25, 2004, which are incorporated by reference in theirentirety for all purposes, both within this section and throughout thespecification.

Other methods of reassortment of influenza virus can optionally mixdilutions of a master donor virus (MDV) and a wild-type virus, e.g., a1:5 dilution of each no matter the concentration of the respectivesolutions, which are then incubated for 24 and 48 hours at 25° C. and33° C. While such an approach is often acceptable for influenza Astrains, influenza B strains do not typically give positive results withsuch protocol. For example, to achieve the proper 6:2 assortment (i.e.,6 genes from the MDV and 2 genes, NA and HA from the wild-type virus)thousands of reassortments must often be done.

Selection and Cloning of Reassortments

The steps in Group 1 also comprise selection of reassorted influenzaviruses. Reassorted influenza A strains are capable of selection ineither cell culture (e.g., CEK cells) or in eggs. However, reassortedinfluenza B strains present problems when reassorted in cell culture(e.g., when selected for in CEK cells). It is believed that CEK cellsinterfere with the M gene in influenza B strains, thus reducing theoverall production. Various methods of vaccine composition production,see, e.g., See, e.g., U.S. patent application Ser. No. 10/788,236 andPCT/US04/05697 both filed Feb. 25, 2004, utilize different steps forvirus reassortment, e.g., selection, such steps can optionally be usedto create virus for the vaccine compositions.

Characterization of Reassortments

Yet other methods of virus/vaccine production utilize applications of ahigh throughput single strand conformation polymorphism/capillaryelectrophoresis (SSCP/CE) assay to determine the gene constellation ofinfluenza viruses used herein. Influenza viruses contain 8 gene segmentsand, as described above, co-infection of a single cell with twodifferent influenza strains can produce reassortant viruses with novelgene constellations distinct from either parent. Thus, some methods canuse a SSCP/CE assay to rapidly determine the gene segment constellationof a large number of influenza virus samples. The influenza viral genesegments are optionally amplified by RT-PCR using fluorescent-labeledprimers specific for each of the eight segments. See, also, Arvin et al.(2000) Clin. Micro. J38(2):839-845 which is incorporated herein byreference for all purposes.

Prevention of Bacterial Contamination

Some methods of virus/vaccine production can comprise steps to detectand/or prevent/detect microbial contamination of eggs in which influenzavirus is produced. The microbial detection strategies of the inventionare useful for rapid/high throughput microbial detection and, thus, aswith many other steps, are useful for increasing throughput andoptionally stability in virus/vaccine production.

Many current influenza vaccine production strategies, which canoptionally be used with the invention herein, use as a component, thetraditional method for influenza virus expansion inspecific-pathogen-free fertile chicken eggs. Possible microbialcontamination can occur in several points in the production of virus ineggs. Unfortunately, the chicken eggs may have some microorganismsoutside of their shells as part of their natural flora. It is alsopossible to have microorganisms enclosed within the shell of the eggduring the development of the chicken embryo. Fertilized chicken eggsare incubated at 37° C. in high humidity for development of the embryo,which constitutes prime incubation conditions for many types ofmicrobial contaminants as well. Another possible time of microbialcontamination occurs when the shell is punctured to inoculate the egg.Even though prior to virus inoculation, the eggs are often sprayed withalcohol, there is still opportunity for microorganisms to enter into theegg.

After expansion of viruses for 2 to 3 days in the eggs, the top of theeggshell is typically removed for manual harvesting of the allantoicfluid containing virus within the egg. See, above. This harvesting isanother point where microbial contamination may originate. Unfortunatelyeggs with such contaminating bioburden may escape detection,necessitating pooling into multiple bottles to minimize the rejection ofthe entire lot due to a failed MPA test. Since three influenza strainsare typically used in vaccine production, blending of the three strainsis required for the final bulk. In-process MPA (microbiological purityassay) testing is performed, e.g., at virus harvest prior to use in theblending and filling to ensure microbial-free product.

After incubation, the “traditional” method of candling is used toidentify infertile and dead eggs that are possibly dead due to naturalcauses or to microbial contamination (i.e., dead eggs may occur due toinfectivity of the virus and/or expansion of microorganisms, both ofwhich require detection and removal of such eggs). Candling comprises,e.g., the process of holding an egg in front of a light source in adarkened room to enable visualization of the developing embryo. Deadeggs are excluded from virus inoculation.

As can be seen from the above points, detection of microbialcontamination can be needed at multiple steps during the manufacture ofinfluenza vaccine. There is a need to eliminate or reduce avian andenvironmental microbes and a need to eliminate or reduce introduction ofenvironmental and human microbes. Current methods for detection ofcontaminating microorganisms include, e.g., compendial methods (MPA andBioburden). Current methods can include, e.g., egg candling during eggpre/post inoculation (which is typically done manually at a rate ofabout 500 eggs/hour/person); MPA and BioBurden tests which are typicallymanual and take about 14 days for MPA and about 3 days for BioBurden(which are done during virus harvest); mycoplasma testing; which istypically done manually and takes about 28 days (done during virusharvest); and mycobacterium testing which is typically manual and takesabout 56 days (done during virus harvest). Again, see, e.g., U.S. patentapplication Ser. No. 10/788,236 and PCT/US04/05697 both filed Feb. 25,2004, for descriptions of various techniques capable of use with thecurrent invention.

Group 2

Aspects of virus/vaccine production that fall into Group 2 includefurther purification and virus expansion, etc. After the process ofcorrect reassortment and cloning of reassortants (i.e., the 6:2viruses), such reassorted virus particles are further purified inembryonated hen eggs and the correct clones are expanded in quantity(again through growth in hen eggs) to generate a master virus strain(MVS) or master virus seed, which, in turn, is further expanded togenerate a master working virus strain (MWVS) or manufacturer's workingvirus seed. Many aspects of purification of virus particles from eggsand use of such purified virus to inoculate more eggs in order to expandthe quantity of virus particles are well known to those skilled in theart. Many such techniques are common in the current production of virusparticles and have been used for at least 40 years. See, e.g., Reimer,et al. Influenza virus purification with the zonal ultracentrifuge,Science 1966, 152:1379-81.

Purification protocols can involve, e.g., ultra-centrifugation insucrose gradients (e.g., 10-40% sucrose), etc. Also, as noted herein,other procedures, etc. listed in other Groups are also optionallypresent within Group 2, e.g., prevention of microbial contamination,etc.

Group 3

Aspects of virus/vaccine production that fall under the heading of Group3 include, e.g., conditioning of the embryonated eggs (e.g., specifichandling and environmental conditions involved in the incubation ofvirus infected eggs) and the harvesting and clarification of influenzavirus from the allantoic fluid of the eggs.

For example, conditioning, washing, candling, and incubating eggs whichcontain the reassorted virus to be used in a vaccine; inoculation,sealing, etc. of such eggs; candling of such eggs; harvesting of thevirus solution (e.g., the allantoic fluid or Viral Harvest (VH)) fromthe eggs; and clarification of the virus solution can all fall withinsuch category. Again, it should be noted that several techniquesapplicable to the steps in Groups 2 are equally applicable to the stepsin Group 3 (e.g., candling, etc.). Several aspects of virus/vaccineproduction that comprise Groups 3 are well known to those skilled in theart. Various aspects of candling of eggs in virus production, as well asinoculation of eggs with viruses and washing, incubating, etc. of sucheggs are well known techniques in the production of virus/vaccines ineggs. Of course, it will be appreciated that such well-known techniquesare used in conjunction with the unique and innovate aspects of thecurrent invention. See, e.g., U.S. patent application Ser. No.10/788,236 and PCT/US04/05697 both filed Feb. 25, 2004, give furthersteps such as rocking, etc. that can also be used with the methods andcompositions of the current invention. Other similar steps can includespecific filtering and warming of compositions, again, see, the same.

Filtering and Warming

The current invention involves aspects of ultra-centrifugation, seeabove, which can fall into the current grouping. In addition, U.S.patent application Ser. No. 10/788,236 and PCT/US04/05697 both filedFeb. 25, 2004 also give other filtering and warming steps that canoptionally be used with the methods and compositions of the currentinvention. As described, the FluMist™ manufacturing process can useembryonated chicken eggs to generate master virus seeds (MVS),manufacturer's working virus seeds (MWVS) and virus harvests (VH). Theseeds and viral harvest may contain bioburden (typically bacterialcontamination), which would cause the seed or bulk virus product lots tobe rejected in the vaccine production process. Of course, it will beappreciated that specific listing or description of particular producttypes used, sizes, etc., is not to be considered limiting on the currentinvention unless specifically stated to be so.

Group 4

Group 4 of the aspects of vaccine formulation/composition productioncomprises, e.g., steps primarily concerned with stabilization (e.g.,through addition of components, alterations in buffer/NAF ratios, etc.)and assays of potency/sterility of virus containing solutions. Thedescription of the current invention above, gives various aspects whichcan optionally be grouped within the current category. See, above.

In some embodiments, the final viral solutions/vaccines comprising liveviruses are stable in liquid form for a period of time sufficient toallow storage “in the field” (e.g., on sale and commercialization whenrefrigerated at 2-8° C., 4° C., 5° C., etc.) throughout an influenzavaccination season (e.g., typically from about September through Marchin the northern hemisphere). Thus, the virus/vaccine compositions aredesired to retain their potency or to lose their potency at anacceptable rate over the storage period. In other embodiments, suchsolutions/vaccines are stable in liquid form at from about 2° C. toabout 8° C., e.g., refrigerator temperature. For example, if a 0.3 logpotency loss was acceptable and the storage period were 9 months, thenan 0.05 log/month decrease in potency would be acceptable. As anotherexample, if a loss of up to 0.75 log were allowed, a rate of less thanor equal to 0.09 log/month would be sufficient to allow stability ofmaterials stored continuously at refrigerator temperature (e.g., 4° C.).In other embodiments, such solutions/vaccines are stable in liquid formwhen stored at from about 2° C. to about 8° C. The stability of thecomposition can comprise between about 1 day and 2 years, between about10 days and about 2 years, between about 20 days and about 2 years,between about 1 month and about 2 years, between about 2 months andabout 2 years, between about 3 months and about 2 years, between about 4months and about 2 years, between about 5 months and about 2 years,between about 6 months and about 2 years, between about 7 months andabout 2 years, between about 8 months and about 2 years, between about 9months and about 2 years, between about 10 months and about 2 years,between about 11 months and about 2 years, between about 12 months andabout 2 years, between about 13 months and about 2 years, between about14 months and about 2 year, between about 15 months and about 2 years,between about 16 months and about 2 years, between about 17 months andabout 2 years, between about 18 months and about 2 years, between about19 months and about 2 years, between about 20 months and about 2 years,between about 21 months and about 2 years, between about 22 months andabout 2 years, between about 23 months and about 2 years, or greaterthan about 2 years.

One may test the stability of a formulation of the invention by a numberof methods. For instance, one may first incubate a formulation atfreezing temperatures (e.g., −25 or −70° C. (+/−10, 20, 30, or 40° C.)and then store (or “incubate”) the formulation at 4-8° C. for a lengthof time. Alternatively, one may test the stability of a formulation ofthe invention by simply storing (or “incubating”) the formulation at4-8° C. for a length of time. Potency could be measured by a number ofmethods as described herein or otherwise known.

Concentration/Diafiltration of Virus Harvests

In some methods of vaccine composition production, virus harvests areoptionally concentrated using an appropriate column. See, U.S. patentapplication Ser. No. 10/788,236 and PCT/US04/05697 both filed Feb. 25,2004.

Stabilizers/Buffers

Vaccine composition production can also optionally include variousdilutions of NAF (typically unfractionated NAF) comprising the virus ofinterest and combinations of, e.g., sucrose, arginine, gelatin, EDTA,etc. See, e.g., U.S. patent application Ser. No. 10/788,236 andPCT/US04/05697, for examples of various combinations possible indifferent vaccine formulations. Such methods and compositions arepreferably stable, e.g., do not show unacceptable losses in potency,e.g., a potency loss of between 0.5-1.0 logs, or less than 0.5 logs, orless than 1.0 logs, over selected time periods (typically at least 6months, at least 9 months, at least 12 months, at least 15 months, atleast 18 months, at least 24 months, etc.) at desired temperatures(e.g., typically 4° C., 5° C., 8° C., from about 2° C. to about 8° C. orgreater than 2° C., etc.).

In some formulations, compositions can comprise a stabilizer of, e.g.,arginine (of pH from about 7.0 to about 7.2), either in combinationwith, or in place of gelatin or gelatin related and/or derived products(e.g., gelatin hydrosylate). See U.S. patent application Ser. No.10/788,236 and PCT/US04/05697. Also, in many virus solutions/vaccinesolutions a base solution of SPG (sucrose, potassium phosphate andmonosodium glutamate) is optionally utilized.

U.S. patent application Ser. No. 10/788,236 and PCT/US04/05697 bothfiled Feb. 25, 2004 give other/additional methods of virus/vaccinecomposition stabilization, e.g., NAF level manipulation, etc.

Definitions

Unless defined otherwise, all scientific and technical terms areunderstood to have the same meaning as commonly used in the art to whichthey pertain. For the purpose of the present invention the followingterms are defined below.

The terms “nucleic acid,” “polynucleotide,” “polynucleotide sequence”and “nucleic acid sequence” refer to single-stranded or double-strandeddeoxyribonucleotide or ribonucleotide polymers, or chimeras or analoguesthereof. As used herein, the term optionally includes polymers ofanalogs of naturally occurring nucleotides having the essential natureof natural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides (e.g.,peptide nucleic acids). Unless otherwise indicated, a particular nucleicacid sequence optionally encompasses complementary sequences, inaddition to the sequence explicitly indicated.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. Thus, genes include coding sequences and/orthe regulatory sequences required for their expression. The term “gene”applies to a specific genomic sequence, as well as to a cDNA or an mRNAencoded by that genomic sequence.

Genes also include non-expressed nucleic acid segments that, forexample, form recognition sequences for other proteins. Non-expressedregulatory sequences include “promoters” and “enhancers,” to whichregulatory proteins such as transcription factors bind, resulting intranscription of adjacent or nearby sequences. A “tissue specific”promoter or enhancer is one that regulates transcription in a specifictissue type or cell type, or types.

The term “vector” refers to the means by which a nucleic acid can bepropagated and/or transferred between organisms, cells, or cellularcomponents. Vectors include plasmids, viruses, bacteriophage,pro-viruses, phagemids, transposons, and artificial chromosomes, and thelike, that replicate autonomously or can integrate into a chromosome ofa host cell. A vector can also be a naked RNA polynucleotide, a nakedDNA polynucleotide, a polynucleotide composed of both DNA and RNA withinthe same strand, a poly-lysine-conjugated DNA or RNA, apeptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like,that are not autonomously replicating. Most commonly, but notnecessarily exclusively, the vectors of herein refer to plasmids.

An “expression vector” is a vector, such as a plasmid that is capable ofpromoting expression, as well as replication of, a nucleic acidincorporated therein. Typically, the nucleic acid to be expressed is“operably linked” to a promoter and/or enhancer, and is subject totranscription regulatory control by the promoter and/or enhancer.

A “bi-directional expression vector” is characterized by two alternativepromoters oriented in the opposite direction relative to a nucleic acidsituated between the two promoters, such that expression can beinitiated in both orientations resulting in, e.g., transcription of bothplus (+) or sense strand, and negative (−) or antisense strand RNAs.

In the context herein, the term “isolated” refers to a biologicalmaterial, such as a nucleic acid or a protein, which is substantiallyfree from components that normally accompany or interact with it in itsnaturally occurring environment. The isolated material optionallycomprises material not found with the material in its naturalenvironment, e.g., a cell. For example, if the material is in itsnatural environment, such as a cell, the material has been placed at alocation in the cell (e.g., genome or genetic element) not native to amaterial found in that environment. For example, a naturally occurringnucleic acid (e.g., a coding sequence, a promoter, an enhancer, etc.)becomes isolated if it is introduced by non-naturally occurring means toa locus of the genome (e.g., a vector, such as a plasmid or virusvector, or amplicon) not native to that nucleic acid. Such nucleic acidsare also referred to as “heterologous” nucleic acids.

The term “recombinant” indicates that the material (e.g., a nucleic acidor protein) has been artificially or synthetically (non-naturally)altered. The alteration can be performed on the material within, orremoved from, its natural environment or state. Specifically, whenreferring to a virus, e.g., an influenza virus, is recombinant when itis produced by the expression of a recombinant nucleic acid.

The term “reassortant,” when referring to a virus, indicates that thevirus includes genetic and/or polypeptide components derived from morethan one parental viral strain or source. For example, a 7:1 reassortantincludes 7 viral genomic segments (or gene segments) derived from afirst parental virus, and a single complementary viral genomic segment,e.g., encoding hemagglutinin or neuraminidase, from a second parentalvirus. A 6:2 reassortant includes 6 genomic segments, most commonly the6 internal genes from a first parental virus, and two complementarysegments, e.g., hemagglutinin and neuraminidase, from a differentparental virus.

The term “introduced” when referring to a heterologous or isolatednucleic acid refers to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid can beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). The term includes suchmethods as “infection,” “transfection,” “transformation,” and“transduction.” In the context of the invention, a variety of methodscan be employed to introduce nucleic acids into prokaryotic cells,including electroporation, calcium phosphate precipitation, lipidmediated transfection (lipofection), etc.

The term “host cell” means a cell that contains a heterologous nucleicacid, such as a vector, and supports the replication and/or expressionof the nucleic acid. Host cells can be prokaryotic cells such as E.coli, or eukaryotic cells such as yeast, insect, amphibian, avian ormammalian cells, including human cells. Exemplary host cells in thecontext of the invention include Vero (African green monkey kidney)cells, BHK (baby hamster kidney) cells, primary chick kidney (PCK)cells, Madin-Darby Canine Kidney (MDCK) cells, Madin-Darby Bovine Kidney(MDBK) cells, 293 cells (e.g., 293T cells), and COS cells (e.g., COS1,COS7 cells).

Influenza Virus

The compositions and methods herein are primarily concerned withproduction of influenza viruses for vaccines. Influenza viruses are madeup of an internal ribonucleoprotein core containing a segmentedsingle-stranded RNA genome and an outer lipoprotein envelope lined by amatrix protein. Influenza A and influenza B viruses each contain eightsegments of single stranded negative sense RNA. The influenza A genomeencodes eleven polypeptides. Segments 1-3 encode three polypeptides,making up a RNA-dependent RNA polymerase. Segment 1 encodes thepolymerase complex protein PB2. The remaining polymerase proteins PB1and PA are encoded by segment 2 and segment 3, respectively. Inaddition, segment 1 of some influenza strains encodes a small protein,PB1-F2, produced from an alternative reading frame within the PB1 codingregion. Segment 4 encodes the hemagglutinin (HA) surface glycoproteininvolved in cell attachment and entry during infection. Segment 5encodes the nucleocapsid nucleoprotein (NP) polypeptide, the majorstructural component associated with viral RNA. Segment 6 encodes aneuraminidase (NA) envelope glycoprotein. Segment 7 encodes two matrixproteins, designated Ml and M2, which are translated from differentiallyspliced mRNAs. Segment 8 encodes NS1 and NS2, two nonstructuralproteins, which are translated from alternatively spliced mRNA variants.

The eight genome segments of influenza B encode 11 proteins. The threelargest genes code for components of the RNA polymerase, PB1, PB2 andPA. Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6encodes the NA protein and the NB protein. Both proteins, NB and NA, aretranslated from overlapping reading frames of a biscistronic mRNA.Segment 7 of influenza B also encodes two proteins: M1 and M2. Thesmallest segment encodes two products, NS1 which is translated from thefull length RNA, and NS2 which is translated from a spliced mRNAvariant.

Influenza Virus Vaccine

Historically, influenza virus vaccines have primarily been produced inembryonated hen eggs using strains of virus selected based on empiricalpredictions of relevant strains. More recently, reassortant viruses havebeen produced that incorporate selected hemagglutinin and neuraminidaseantigens in the context of an approved attenuated, temperature sensitivemaster strain. Following culture of the virus through multiple passagesin hen eggs, influenza viruses are recovered and, optionally,inactivated, e.g., using formaldehyde and/or β-propiolactone (oralternatively used in live attenuated vaccines).

However, production of influenza vaccine in this manner has severalsignificant concerns. For example, contaminants remaining from the heneggs can be highly antigenic and/or pyrogenic, and can frequently resultin significant side effects upon administration. Thus, another methodinvolves replacement of some percentage of egg components with animalfree media. More importantly, virus strains designated for vaccineproduction must be selected and distributed, typically months in advanceof the next flu season to allow time for production and inactivation ofinfluenza vaccine. Again, any improvements in stability in storage timeand/or of storage at a more convenient temperature (e.g., refrigeratortemperature of about 2-8° C., e.g., as through use of the methods andcompositions of the current invention), are thus quite desirable.

Attempts at producing recombinant and reassortant vaccines in cellculture have been hampered by the inability of some of the strainsapproved for vaccine production to grow efficiently under standard cellculture conditions. Thus, prior work by the inventor and his coworkersprovided a vector system, and methods for producing recombinant andreassortant viruses in culture, thus, making it possible to rapidlyproduce vaccines corresponding to one or many selected antigenic strainsof virus. See, e.g., U.S. patent application No. 60/375,675 filed Apr.26, 2002, PCT/US03/12728 filed Apr. 25, 2003 and U.S. Ser. No.10/423,828 filed Apr. 25, 2003, PCT/US05/017734 filed May 20, 2005. Ofcourse, such reassortments are optionally further amplified in hen eggs.Typically, the cultures are maintained in a system, such as a cellculture incubator, under controlled humidity and CO₂, at constanttemperature using a temperature regulator, such as a thermostat toinsure that the temperature does not exceed 35° C. Such pioneering work,as well as other vaccine production, can be further optimized throughuse of the current invention in whole or part.

Reassortant influenza viruses can be readily obtained by introducing asubset of vectors corresponding to genomic segments of a masterinfluenza virus, in combination with complementary segments derived fromstrains of interest (e.g., antigenic variants of interest). Typically,the master strains are selected on the basis of desirable propertiesrelevant to vaccine administration. For example, for vaccine production,e.g., for production of a live attenuated vaccine, the master donorvirus strain may be selected for an attenuated phenotype, coldadaptation and/or temperature sensitivity.

FluMist®

As mentioned previously, numerous examples and types of influenzavaccine exist. An exemplary influenza vaccine is FluMist which is alive, attenuated vaccine that protects children and adults frominfluenza illness (Belshe et al. (1998) The efficacy of live attenuated,cold-adapted, trivalent, intranasal influenza virus vaccine in childrenN. Engl. J. Med. 338:1405-12; Nichol et al. (1999) Effectiveness oflive, attenuated intranasal influenza virus vaccine in healthy, workingadults: a randomized controlled trial JAMA 282:137-44). In typicalembodiments, the methods and compositions of the current invention arepreferably adapted to, or used with, production of FluMist vaccine.However, it will be appreciated by those skilled in the art that thesteps/compositions herein are also adaptable to production of similar oreven different viral vaccines and their compositions.

FluMist™ vaccine strains contain, e.g., HA and NA gene segments derivedfrom the wild-type strains to which the vaccine is addressed along withsix gene segments, PB1, PB2, PA, NP, M and NS, from a common masterdonor virus (MDV). The MDV for influenza A strains of FluMist (MDV-A),was created by serial passage of the wild-type A/Ann Arbor/6/60(A/AA/6/60) strain in primary chicken kidney tissue culture atsuccessively lower temperatures (Maassab (1967) Adaptation and growthcharacteristics of influenza virus at 25 degrees C. Nature 213:612-4).MDV-A replicates efficiently at 25° C. (ca, cold adapted), but itsgrowth is restricted at 38 and 39° C. (ts, temperature sensitive).Additionally, this virus does not replicate in the lungs of infectedferrets (att, attenuation). The ts phenotype is believed to contributeto the attenuation of the vaccine in humans by restricting itsreplication in all but the coolest regions of the respiratory tract. Thestability of this property has been demonstrated in animal models andclinical studies. In contrast to the ts phenotype of influenza strainscreated by chemical mutagenesis, the ts property of MDV-A does notrevert following passage through infected hamsters or in shed isolatesfrom children (for a recent review, see Murphy & Coelingh (2002)Principles underlying the development and use of live attenuatedcold-adapted influenza A and B virus vaccines Viral Immunol.15:295-323).

Clinical studies in over 20,000 adults and children involving 12separate 6:2 reassortant strains have shown that these vaccines areattenuated, safe and efficacious (Belshe et al. (1998) The efficacy oflive attenuated, cold-adapted, trivalent, intranasal influenza virusvaccine in children N. Engl. J. Med. 338:1405-12; Boyce et al. (2000)Safety and immunogenicity of adjuvanted and unadjuvanted subunitinfluenza vaccines administered intranasally to healthy adults Vaccine19:217-26; Edwards et al. (1994) A randomized controlled trial of coldadapted and inactivated vaccines for the prevention of influenza Adisease J. Infect. Dis. 169:68-76; Nichol et al. (1999) Effectiveness oflive, attenuated intranasal influenza virus vaccine in healthy, workingadults: a randomized controlled trial JAMA 282:137-44). Reassortantscarrying the six internal genes of MDV-A and the two HA and NA genesegments of a wild-type virus (i.e., a 6:2 reassortant) consistentlymaintain ca, is and att phenotypes (Maassab et al. (1982) Evaluation ofa cold-recombinant influenza virus vaccine in ferrets J. Infect. Dis.146:780-900). Production of such reassorted virus using B strains ofinfluenza is more difficult, however.

Recent work, see, see, e.g., U.S. patent application No. 60/375,675filed Apr. 26, 2002, PCT/US03/12728 filed Apr. 25, 2003 and U.S. Ser.No. 10/423,828 filed Apr. 25, 2003, PCT/US05/017734 filed May 20, 2005has shown an eight plasmid system for the generation of influenza Bvirus entirely from cloned cDNA, and methods for the production ofattenuated live influenza A and B virus suitable for vaccineformulations, such as live virus vaccine formulations useful forintranasal administration.

The system and methods described previously are useful for the rapidproduction in cell culture of recombinant and reassortant influenza Aand B viruses, including viruses suitable for use as vaccines, includinglive attenuated vaccines, such as vaccines suitable for intranasaladministration such as FluMist®. The methods of the current inventionherein, are optionally used in conjunction with or in combination withsuch previous work involving, e.g., reassorted influenza viruses forvaccine production to produce viruses for vaccines in a more stable,consistent and productive manner.

Cell Culture

As previously stated, influenza virus optionally can be grown in cellculture. Typically, propagation of the virus is accomplished in themedia compositions in which the host cell is commonly cultured. Suitablehost cells for the replication of influenza virus include, e.g., Verocells, BHK cells, MDCK cells, 293 cells and COS cells, including 293Tcells, COS7 cells. Commonly, co-cultures including two of the above celllines, e.g., MDCK cells and either 293T or COS cells are employed at aratio, e.g., of 1:1, to improve replication efficiency. Typically, cellsare cultured in a standard commercial culture medium, such as Dulbecco'smodified Eagle's medium supplemented with serum (e.g., 10% fetal bovineserum), or in serum free medium, under controlled humidity and CO₂concentration suitable for maintaining neutral buffered pH (e.g., at pHbetween 7.0 and 7.2). Optionally, the medium contains antibiotics toprevent bacterial growth, e.g., penicillin, streptomycin, etc., and/oradditional nutrients, such as L-glutamine, sodium pyruvate,non-essential amino acids, additional supplements to promote favorablegrowth characteristics, e.g., trypsin, β-mercaptoethanol, and the like.

Procedures for maintaining mammalian cells in culture have beenextensively reported, and are well known to those of skill in the art.General protocols are provided, e.g., in Freshney (1983) Culture ofAnimal Cells: Manual of Basic Technique, Alan R. Liss, New York; Paul(1975) Cell and Tissue Culture, 5^(th) ed., Livingston, Edinburgh; Adams(1980) Laboratory Techniques in Biochemistry and Molecular Biology-CellCulture for Biochemists, Work and Burdon (eds.) Elsevier, Amsterdam.Additional details regarding tissue culture procedures of particularinterest in the production of influenza virus in vitro include, e.g.,Merten et al. (1996) Production of influenza virus in cell cultures forvaccine preparation in Cohen and Shafferman (eds.) Novel Strategies inDesign and Production of Vaccines, which is incorporated herein in itsentirety for all purposes. Additionally, variations in such proceduresadapted to the present invention are readily determined through routineexperimentation and will be familiar to those skilled in the art.

In a specific embodiment of the invention, host cells of the inventionare cultured and/or infected under serum-free conditions, in thepresence or absence of trypsin, and are cultured and/or infected attemperatures ranging from 30 degrees Celsius to 39 degrees Celsius; or30 degrees Celsius, or 31 degrees Celsius, 32 degrees Celsius, 33degrees Celsius, 34 degrees Celsius, 35 degrees Celsius, 36 degreesCelsius, 37 degrees Celsius, 38 degrees Celsius, or 39 degrees Celsius.

Cells for production of influenza virus can be cultured in serumcontaining or serum free medium. In some cases, e.g., for thepreparation of purified viruses, it is typically desirable to grow thehost cells in serum free conditions. Cells can be cultured in smallscale, e.g., less than 25 ml medium, culture tubes or flasks or in largeflasks with agitation, in rotator bottles, or on microcarrier beads(e.g., DEAE-Dextran microcarrier beads, such as Dormacell, Pfeifer &Langen; Superbead, Flow Laboratories; styrene copolymer-tri-methylaminebeads, such as Hillex, SoloHill, Ann Arbor) in flasks, bottles orreactor cultures. Microcarrier beads are small spheres (in the range of100-200 microns in diameter) that provide a large surface area foradherent cell growth per volume of cell culture. For example a singleliter of medium can include more than 20 million microcarrier beadsproviding greater than 8000 square centimeters of growth surface. Forcommercial production of viruses, e.g., for vaccine production, it isoften desirable to culture the cells in a bioreactor or fermenter.Bioreactors are available in volumes from under 1 liter to in excess of100 liters, e.g., Cyto3 Bioreactor (Osmonics, Minnetonka, Minn.); NBSbioreactors (New Brunswick Scientific, Edison, N.J.); laboratory andcommercial scale bioreactors from B. Braun Biotech International (B.Braun Biotech, Melsungen, Germany).

Regardless of the culture volume, in many desired aspects of the currentinvention, it is important that the cultures be maintained at anappropriate temperature, to insure efficient recovery of recombinantand/or reassortant influenza virus using temperature dependent multiplasmid systems (see, e.g., Multi-Plasmid System for the Production ofInfluenza Virus, cited above), heating of virus solutions forfiltration, etc. Typically, a regulator, e.g., a thermostat, or otherdevice for sensing and maintaining the temperature of the cell culturesystem and/or other solution, is employed to insure that the temperatureis at the correct level during the appropriate period (e.g., virusreplication, etc.).

In some methods (e.g., wherein reassorted viruses are to be producedfrom segments on vectors) vectors comprising influenza genome segmentsare introduced (e.g., transfected) into host cells according to methodswell known in the art for introducing heterologous nucleic acids intoeukaryotic cells, including, e.g., calcium phosphate co-precipitation,electroporation, microinjection, lipofection, and transfection employingpolyamine transfection reagents. For example, vectors, e.g., plasmids,can be transfected into host cells, such as COS cells, 293T cells orcombinations of COS or 293T cells and MDCK cells, using the polyaminetransfection reagent TransIT-LT1 (Mirus) according to the manufacturer'sinstructions in order to produce reassorted viruses, etc. Approximately1 μg of each vector to be introduced into the population of host cellswith approximately 2 μl of TransIT-LT1 diluted in 160 μl medium,preferably serum-free medium, in a total volume of 200 μl. TheDNA:transfection reagent mixtures are incubated at room temperature for45 minutes followed by addition of 800 μl of medium. The transfectionmixture is added to the host cells, and the cells are cultured asdescribed above or via other methods well known to those skilled in theart. Accordingly, for the production of recombinant or reassortantviruses in cell culture, vectors incorporating each of the 8 genomesegments, (PB2, PB1, PA, NP, M, NS, HA and NA) are mixed withapproximately 20 μl TransIT-LT1 and transfected into host cells.Optionally, serum-containing medium is replaced prior to transfectionwith serum-free medium, e.g., Opti-MEM I, and incubated for 4-6 hours.

Alternatively, electroporation can be employed to introduce such vectorsincorporating influenza genome segments into host cells. For example,plasmid vectors incorporating an influenza A or influenza B virus arefavorably introduced into Vero cells using electroporation according tothe following procedure. In brief, approximately 5×10⁶ Vero cells, e.g.,grown in Modified Eagle's Medium (MEM) supplemented with 10% FetalBovine Serum (FBS) are resuspended in 0.4 ml OptiMEM and placed in anelectroporation cuvette. Twenty micrograms of DNA in a volume of up to25 μl is added to the cells in the cuvette, which is then mixed gentlyby tapping. Electroporation is performed according to the manufacturer'sinstructions (e.g., BioRad Gene Pulser II with Capacitance Extender Plusconnected) at 300 volts, 950 microFarads with a time constant of between28-33 msec. The cells are remixed by gently tapping and, approximately1-2 minutes following electroporation, 0.7 ml MEM with 10% FBS is addeddirectly to the cuvette. The cells are then transferred to two wells ofa standard 6 well tissue culture dish containing 2 ml MEM, 10% FBS. Thecuvette is washed to recover any remaining cells and the wash suspensionis divided between the two wells. Final volume is approximately 3.5 mL.The cells are then incubated under conditions permissive for viralgrowth, e.g., at approximately 33° C. for cold adapted strains. See,e.g., US20050158342, which is incorporated by reference herein.

Kits

To facilitate use of the methods and compositions of the invention, anyof the vaccine components and/or compositions, e.g., virus in variousformulations, etc., and additional components, such as, buffer, cells,culture medium, useful for packaging and infection of influenza virusesfor experimental or therapeutic vaccine purposes, can be packaged in theform of a kit. Typically, the kit contains, in addition to the abovecomponents, additional materials which can include, e.g., instructionsfor performing the methods of the invention, packaging material, and acontainer.

EXAMPLE 1

Development of Liquid FluMist

A total of 19 monovalent bulk lots of virus were initiated underprotocol, including four development lots (CB0006H, CB0008H, CG0017H,CB0018H and CB0019H). Lots CB0018H and CB0019H were conducted to supplymaterial for various studies and are not discussed further in thisapplication. Two lots (CB00180H and CB0012H) were combined after theultra-centrifugation step to produce A/Sydney monovalent bulk CB0020H.Each lot was initiated with about 2000 eggs.

Egg handling and incubation conditions were set up similar to theprocess used for production of frozen FluMist, however, manualinoculation and harvesting were used due to the smaller scale, etc. Theultracentrifuge process was scaled down to smaller equipment made by thesame manufacturer (Discovery 90, made by Hitachi and marketed bySorvall/heraus), with a Model P32CT rotor having approximately 470 mL oftotal capacity. Clarified, stabilized virus harvest was loaded onto a20% to 60% sucrose gradient, banded for one hour at 32,000 rpm, andeluted into 20 mL fractions. Fractions containing peak HA levels werepooled, diluted to 0.2M sucrose concentration, filtered through a 0.2micron filter, and then transferred into 250 mL flexible polymer bags(Stedim) which were frozen and held below −60° C. for furthermanufacture.

After a short series of test runs, cGMP manufacturing was initiated tosupport further clinical testing of liquid FluMist. A/Beijing/262/95(H1N1) and A/Sydney/05/97 (H3N2) were again manufactured, and the Bstrain was changed to B/Yamanashi/166/98. Monovalent bulks produced werefrozen and shipped for blending, filing, and packaging. Of course, itwill be appreciated here and throughout, that use of particular strains(e.g., Beijing, etc.) should not necessarily be taken as limiting unlessspecifically stated to be so. Thus for example, the methods andformulations herein can optionally use different strains each influenzaseason to produce different RTS compositions, etc.

Development and Clinical Trials

The process for liquid compositions was developed and includedmonovalent bulk lots CAZ001-024 and CAZ035-043. The clinical trialmaterial (CTM) included monovalent bulk lots CAZ025-CAZ034.

The liquid FluMist manufacturing process developed and used for CTM-1 isdivided into six discrete process stages below. The process stages 1through 5 were defined to match the major process steps as conductedusing separate manufacturing instruction documents. Stage 6 includes theentire blend and fill process. It will be appreciated that such stagescan be roughly compared to the generalized steps outlined below formanufacturing/production of vaccine compositions in general.

Stages

Stage 1: receipt and sanitization of SPF eggs; Stage 2: production ofvirus harvest; Stage 3: concentration of virus by zonal centrifugation;Stage 4: pooling and dilution of peak fractions; Stage 5: sterilefiltration and monovalent bulk storage; and, Stage 6: blend and fill

A process flow diagram for the CTM-1 monovalent bulk production is shownin FIG. 1. The blend and fill process flow is shown in FIG. 2.

Receipt and Sanitization of SPF eggs

Specific pathogen-free (SPF) embryonated eggs were purchased fromSPAFAS, Inc. which utilizes flock management and surveillance programsdesigned to provide suitable assurance of disease-free eggs. Eggs werelaid in the United States and sanitized using Clorwash and Quat 800sprays. The eggs were then transported by air and road using packagingsufficient to maintain cleanliness and avoid freezing or overheating.Upon receipt, the cold fertile eggs were inspected and stored in an SPFincubation unit at 14° C. +/−2° C. with 60-80% relative humidity (RH)for a maximum of 7 days. The eggs were transferred to trays from thecartons supplied by the vendor and a batch number assigned. Eggs wereplaced on 36-egg Jamesway trays and the eggshell surfaces sanitized withChlorwash and Quat 800 in an automated egg sanitization system tominimize bioburden prior to placing them in the incubator. Chlorwash wasprepared at a range of 43-44° C. and 48-49° C. and Quat 800 was preparedat a range of 48-49° C. Following sanitization, the eggs were placed ontrolleys and air dried at ambient temperature for not less than 2 hours.The eggs were then placed in a Buckeye Incubator and incubated at 37.5°C. +/−1° C. with 60-80% RH for 264 hours +/−12 hours. After incubation,the SPF eggs were transferred from the SPF egg unit to a candling area.Using a fiber optic lamp, the position of the air sac was located ineach egg. Dead or infertile eggs were discarded, and the fertile eggsmarked for inoculation.

Production of Virus Harvests

The fertile eggs were surface sanitized with 70% industrial methylatedspirits (IMS) prior to inoculation and then transferred for massinoculation. In preparation of the diluted manufacturer's working virusseed (MWVS), the master virus seeds used to produce the MWVS for phase 1clinical trials were produced using the classical reassortment method.The MWVS, was transferred into the AVU and serial dilutions of thethawed MWVS were prepared in sterile 0.01M phosphate buffered saline, pH7.7 within a microbiological safety cabinet using a sterile disposablepipette for each transfer. The inoculation occurred under laminar flowin a class 10,000 room using a semi-automatic Bibby inoculation machine,which penetrates and inoculates 36 eggs per tray. The target titer ofthe inoculum was log₁₀ 2.1 TCID₅₀ per 0.1 mL and the dilution wascalculated from the predetermined titer of the MWVS. Preparation of eachvirus inoculum was completed within 2 hours of the removal of the vialfrom the freezer. Aliquots of the inoculum were stored at 5+/−3° C.until use. Inoculum was used within 8 hours of preparation.

Inoculation of SPF Eggs

Each tray of eggs was hand-fed into the Bibby automated inoculationmachine. A punch was used to pierce the eggshell and penetrate the eggto a preset adjustable depth. The inoculation needles extended into theegg and the dosing pump delivered 0.1 mL of the MWVS inoculum into eachegg after which the punch was withdrawn. Following inoculation, the eggtray was removed by an operator and the process repeated with a new trayof eggs. The CAIV inoculated eggs were incubated at 33+/−1° C. for atime determined by the growth curve of the virus strain. Afterincubation, the SPF embryonated eggs were chilled to 28° C. for 18+/−6hours. At the end of chilling, the eggs were transferred for theharvesting step.

Concentration of Virus by Zonal Centrifugation

Virus Harvest (VH)

Eggs were harvested with an automatic harvest machine (Bibby). The trayswere hand fed to a decapitation station where the upper section of theeggshell was removed to create an access hole for harvest needle entry.The eggs were visually inspected prior to harvest and unsuitable eggs(e.g., discolored) rejected. The acceptable eggs proceeded to a harveststation, where the allantoic fluid was withdrawn using needles andsuction provided by a vacuum system. The harvest was collected mixed ina 100 L jacketed stainless vessel with a jacket temperature of 2-8° C.Samples of the virus harvest were collected from the VH pool and testedfor: potency (TCID₅₀), safety, avian leucosis, M. tuberculosis,mycoplasma, extraneous virus, identity, reverse transcriptase assay,virus genotype, virus phenotype, and attenuation.

Stabilization and Clarification

The pooled virus harvest (VH) was immediately stabilized at 2-8° C. to afinal concentration of 0.2 M sucrose, 0.01M phosphate, 0.005M glutamate(SPG), with 10× SPG (9 parts VH to 1 part 10× SPG). Samples werecollected for potency and bioburden prior to clarification. Thestabilized virus harvest was clarified by 5 μm depth filtration toremove particulates prior to purification by high-speed continuous flowcentrifugation. Clarified virus harvest was sampled for potency.

Zonal Centrifugation, Collection, and HA Assay of Peak Fractions

Prior to use, the centrifuge rotor was sanitized with a 1:20 dilution ofconcentrated formaldehyde solution. The clarified VH was loaded onto asucrose gradient (10-60% sucrose in phosphate buffer, pH 7.2) forcontinuous high-speed centrifugation using a Hitachi CP40Y zonalcentrifuge. The gradient was formed by addition to the centrifuge of a60% sucrose solution followed by a 10% sucrose solution in phosphatebuffer. The centrifuge speed was set to 4,000 rpm for 20 minutes toallow the sucrose solutions to form a density gradient. Centrifugetemperature was set at 2-8° C. during gradient formation. Followinggradient formation, buffer flow was initiated and the rotor speed wasincreased to 40,000 rpm, before the clarified VH was loaded onto thegradient at a rate of 20L per hour at 40,000 rpm. For a typical CTM-1batch with 10,000 eggs harvested and 6 mL/egg, the duration of theloading step was about three hours. Following loading of the clarifiedVH, centrifugation was continued at 40,000 rpm for an additional hour toallow the virus to band. The virus particles migrated to the 38-45%sucrose portion of the gradient and concentrate into a “band.” At theend of this step, the centrifuge was gradually slowed and then stoppedto allow collection of the virus peaks. 100 mL fractions were collectedunder laminar flow into 125 mL sterile polycarbonate bottles. Fractionswere held at 2-8° C. for approximately 1 hour while fractions wereassayed for HA activity. Peak fractions were typically found between38-45% of the sucrose gradient.

Pooling and Dilution of Peak Fractions

The centrifuge peak fractions, as identified by the HA assay, werepooled under a laminar flow hood by aseptically pouring the fractionsinto a 5 L sterile glass bottle, and mixed by swirling. The refractiveindex (RI) was read to determine sucrose concentration and the poolsampled for potency. The centrifuge peak fraction pool (CP) was dilutedby aseptically adding a calculated volume of sterile cold (2-8° C.)phosphate-glutamate buffer, pH 7.2 (PBG buffer) to a final concentrationof 0.2M sucrose, 0.1M phosphate, and 0.005 M glutamate. This wastypically a 1:6 dilution. The diluted centrifuge peak fraction pool(DCP) was sampled for potency and bioburden.

Sterile Filtration and Monovalent Bulk Storage

The DCP was pumped through sterile tubing to a Class 100 filling roomfor sterile filtration. The DCP was sterile filtered with a 0.22 μmfilter into a sterile 5L glass bottle under a Laminair Air Flow hood.The filtered monovalent bulk was mixed and sampled for potency,identity, and sterility testing. The 0.22 μm filter was integrity testedbefore and after the filtration process. The MB was then aliquoted intosterile one liter polycarbonate bottles. The MB was collected in 500 mLaliquots with a total volume of three to four liters. The sterileone-liter PC bottles were stored at −60° C. or below. The titer of thebulk virus was determined by TCID₅₀ assay. Monovalent bulk was thenshipped at ≦−60° C. for blending and testing.

Blend and Fill

The major steps for the blend and fill process of the preparation of thebulk trivalent blend were: Thawing, Preparation of Diluent, Blending,Filling, and Packaging.

Thawing and Blending

The appropriate 1 bottles of monovalent bulk were removed from frozenstorage (≦−60° C. for MB) and transferred to a thaw room. The requiredquantities of the three individual monovalent bulks and SPG diluent werecalculated based on the bulk infectivity titer and the requiredformulation strength. The bottles of MB were loaded into a 33±3° C.water bath and manually agitated every five minutes. Thawing wasmonitored visually to ensure that all bottles were thawed before leavingthe water bath, and that all thawed bottles were removed every 15minutes. Once thawed, the bottles of MB were moved to a 5±3° C.refrigerator and held until all of the required bottles had been thawed.

Sterile sucrose phosphate glutamate (SPG) diluent was manufactured byBioWhittaker (Walkersville, Md.). Just prior to manufacture of theliquid FluMist blend, about one-third of the amount of SPG diluentrequired for the blend was added to a sterile 2-L bottle. The bottle waswarmed to room temperature, then hydrolyzed porcine gelatin (powder) wasadded in the amount need to reach a level of 10 mg/ml in the finalblend. The SPG diluent was then passed through the filter (washingthrough any residual gelatin) to reach the target volume specified inthe blend calculation and stored at 5±3° C. until use.

Thawed bottles were moved to a blend room and virus asepticallytransferred to a 5 L glass process vessel. The vessel was kept at 5±3°C. throughout the blending process and the subsequent filling process byusing refrigerated cold packs. The SPG-gelatin diluent was added afterthe three virus strains had been added, and, if necessary, the pH wasadjusted with HCl to 7.2±0.3. The three virus strains and diluent wereblended by continuous mixing with a magnetic stir bar and stir plate.

Filling and Packaging

Following pH adjustment, the bulk trivalent blend vessel was moved to afilling room and connected to an INOVA filler. The INOVA filling machinefilled a preset volume of product into a row of eight BD HYPAKdisposable sprayers and then stoppered the sprayers. Sprayer filling andstoppering operations and weight check verifications continued asdirected. A new tub of sprayers was manually placed at the in-feedstations upon completion of each fill cycle and the tub of filledsprayers removed to the discharge station. The blend vessel wasmaintained at 5±3° C. with cold packs during the filling process.

Following filling of the nasal sprayers, the filled trivalent vaccineswere loaded into tubs and immediately transported via cart to apackaging area for final packaging and labeling. Packaged and labeledsprayers were stored at 5±3° C.

Optimization of Upstream Operating Parameters

Clarification of Harvest by Filtration (5 μm)

Filtration vs. Low Speed Centrifugation: Clarification potency lossesusing the standard low-speed centrifugation for frozen FluMist aretypically estimated at 0.2 to 0.3 log₁₀ TCID₅₀/mL. An estimatedcentrifugation loss for two strains was: titer loss was negligible forone strain and 0.3 logs (59% step yield) for a second strain using thestandard conditions of 3400 g for 20 minutes. When a 5 micron depthfilter was used as an alternative to centrifugation during pre-CTMdevelopment runs, the average clarification step yield was estimated at41%. When TCID₅₀ assay variability was taken into consideration, it wasconcluded that either method of clarification provided better resultsthan adding after clarification. Filtration was chosen as theclarification method based on ease of operation and scalability.

Comparison of Depth Filter Pore Sizes: Development lots CAZ015-CAZ017plus the ten CTM-1 lots CAZ025 through CAZ034 were used to estimatedepth filtration losses using the CTM-1 process. A/Beijing, A/Sydney,and B/Harbin have clarification step yields of 147%, 78%, and 73%respectively for the 5 μm clarification step.

The 5 μm (Pall Profile II) filter was compared to a 20 μm depth filter(Pall Profile Star), which was used for lots CAZ035 (A/Sydney), CAZ036(A/Beijing) and CAZ037 (B/Harbin). A/Beijing, A/Sydney, and B/Harbin hadclarification step yields of 65%, 35%, and 209% respectively for the 20μm clarification step. These yields were comparable to 5 μmclarification results, however a process change to the 20 μm filter wasnot recommended for all embodiments due to significant contamination ofclarified harvests by red blood cells when larger pore size was used.

Optimization of Downstream Operating Parameters:

Centrifuge Scale

Centrifuge loading and temperature studies were performed using thelarge-scale CP40Y centrifuge and RP40CT Type D continuous-flow rotor;these compared various egg batch sizes and centrifuge temperature setpoints (see FIG. 3). CA0Z15 (B/Harbin), CAZ018 (A/Sydney), CAZ020(A/Beijing) and CAZ022 (A/Sydney) had batch sizes of 10 K eggs and acentrifuge set temperature of 4° C. CAZ 016 (B/Harbin), CAZ019(A/Sydney), and CAZ021 (A/Beijing) had batch sizes of 20 K eggs and acentrifuge set temperature of 14° C. A loading flow rate of 20L/hr wasused for all of these studies. Percent recovery in the dilutedpeak-fraction pool (step yield) was based on the clarified harvest titerin the figure. As can be seen, lots with 10 K egg batches had a recoveryrange from 40-184% whereas the 20 K egg batches ranged from 40-55%. Theaverage recovery for the 10 K batch size was 113% and the 20 K egg batchrecoveries averaged 46%. A batch size of 10,000 eggs per run wasselected for CTM manufacturing as a conservative limit in this step ofthe development process.

Temperature

Development runs CAZ015 through CAZ022 were performed usingultracentrifuge rotor set point temperature either at 4° C. or 14° C.With one exception, the development runs at 14° C. were also run atlarger scale than typical (20,000 eggs per batch). In general thesebatches had lower recoveries of live virus, however this could be due tocentrifuge loading rather that temperature effects. In view of thebetter results at 4° C. and the possibility of increased microbialgrowth rates at higher temperatures, a temperature of 4° C. was selectedas the ultracentrifuge temperature set point at this point in thedevelopment of the process.

Clinical Trial 1 (CTM-1)

The monovalent virus lots for CTM-1 were produced and includedmonovalent bulk lots CAZ025 through CAZ034. In-process QC assay resultsfor the CTM-1 monovalent bulk lots are presented in FIG. 3 and herein.Average step yields per strain are presented in FIG. 3 and herein.Results are summarized below.

Potency: The potencies (log₁₀ TCID₅₀ mL) of B/Harbin and A/Beijing virusharvest lots were equal or greater than 8.1. A/Sydney ranged from 7.5 to8.8 except for CAZ030, which showed very low titer (6.0) because of yolkcontamination in the harvest. The titer of the monovalent bulk forA/Beijing was 9.3 or above, for B/Harbin the titer ranged from 8.4 to9.85 and for A/Sydney ranged from 7.9 to 8.6 (excluding CAZ030). ForCAZ030 the titer was below the acceptable level for blending asdiscussed below.

Process Yield: Process yields for each lot are referenced in the Figuresand discussed further herein. Process yields were not corrected forsampling losses. A/Beijing had an average overall process yield of 16%(163 doses/egg), A/Sydney had an average yield of 13% (6 doses/egg), andB/Harbin had an average yield of 93% (86 doses/egg). See figures. Theerratic yield estimates reflect assay variability as well as the actualprocess performance. In particular, the B/Harbin results were skewedupward by a very high estimate of Lot CAZ028 monovalent bulk titer(resulting in estimated 252% process yield and 246 doses/egg). Thediluted peak pool titer in this case was determined at 0.95 logs higherthan the centrifuge peak pool, though a decline would be expected due todilution. Likewise the filtered monovalent bulk titer was higher thanthe prefiltered material, which can be accounted for by assayvariability. The other two B/Harbin lots, (CAZ026 and CAZ027) hadprocess yields of 20% and 8% respectively, and an egg yield of 7doses/egg for both lots. The trivalent blends that included bulk lotCAZ028 (CBF1004 and CBF1007) resulted in B/Harbin potencies in finalproduct sprayers that were 0.6 and 0.4 log₁₀ TCID₅₀ per dose. Thisfurther suggests that the CAZ028 monovalent bulk titer wasoverestimated.

Bioburden and Endotoxin: Virus harvest, stabilized harvest and dilutedcentrifuge peak fractions were tested for bioburden. Bioburden testresults do not give a clear picture of organism loads through theprocess: test results on virus harvest and diluted centrifuge peakfraction pool samples consisted primarily of “none detected” (eightreadings); or “>100 cfu/mL” (ten readings) with only two intermediatevalues. Endotoxin values for the monovalent bulks varied from <5 EU/mLto 587 EU/mL.

Sucrose Concentration: The centrifuge peak fraction pools had sucroseconcentrations ranging from 39 to 43.5% (CAZ030 was not included in therange). The monovalent bulks had an average sucrose concentration of7.6%.

The monovalent bulk release assay results are presented in the figures.All of the CTM-1 lots except for CAZ030 (which was not tested) passedthe bulk release assays.

SDS-PAGE Analysis: Monovalent bulks were further characterized by 10%SDS-PAGE gels stained with Gel Code Blue. Based on the calculatedprotein molecular weights and number of copies per particle forinfluenza A and B strains, gel bands in the region of 50-60 kDa(HA1protein), 56 kDa (NP protein) and 27 kDa (M1protein) would beexpected to be prominent in gels of purified influenza virus. Ha2protein (23 to 30 kD) may also be present.

SDS-PAGE gels for A/Beijing/262/95 CAZ031, and 33 and A/Sydney/05/97CAZ029, 30, and 34 were compared. The range of resolution for the 10%gels was nominally 200-31 kDa. Because of this limitation,interpretation of HA1 protein (23-30 kDa) and M protein bands (21-27kDA) should be made with caution.

For A/Beijing and A/Sydney monovalent bulk (MB) lots, a dark band wasobserved in the expected region of NP protein (just above the 55 kDamarker) for the A/Beijing samples, and a band was seen just above 116kDa for both strains. Both strains contained a band near the gel front(below 31 kD) consistent with M protein. The HA1 band expected at orjust above the 66 kD marker was less strong, possibly due toheterogeneity in glycoprotein molecular weight resulting in a moredispersed banding pattern. Staining of the putative NP1 protein alsoseems to be diminished. For the A/Sydney samples a wider variety ofbands were observed, consistent with a greater proportion of egg proteinin the monovalent bulk.

Doses per Lot and Egg: The dose calculation for all three strains wasbased on a 0.24 mL average fill volume containing 1.2 E+07 TCID₅₀particles. Based on the dose calculation above, the A/Beijing monovalentlots had an average of 1,576,022 total doses per lot. Average totaldoses per lot for B/Harbin and A/Sydney were 601,446 and 50,825 dosesper lot respectively, or 38% and 3.2% of the average A/Beijing totaldoses per lot respectively. These results translate to the followingvalues for monovalent doses per egg: 163 doses/egg for A/Beijing, 86doses/egg for B/Harbin-like and 6 doses/egg for A/Sydney. Thecalculation for doses/egg was based on the total number of eggsharvested to manufacture the lot.

Summary of Egg Yields: A/Sydney had the highest average harvest volumeper egg yield, followed by A/Beijing and B/Harbin. The yield per eggaveraged 5.6 mL/egg for B/Harbin, 6.5 mL/egg for A/Beijing, and 6.8mL/egg for A/Sydney. The overall yield of viable eggs for productionaveraged 82%. Egg yields were based on the total number of eggsharvested compared to total number of eggs for production. Percent eggsrejected pre-inoculation averaged 10%. Percent rejection post-incubationaveraged 5%, with the exception of CAZ028, which had a 29% rejectionrate.

Purification Yield Summary (TCID₅₀): Clarified harvest potency valueswere higher than the starting material for some of the lots, resultingin step yields greater than 100%. Step yield estimates are affected bythe variability of the TCID₅₀ assay, which had a standard deviationabove 0.3 log₁₀ TCID₅₀/mL as performed at the time of the productionruns. In addition, the step yield at the Diluted Centrifuge Pak FractionPool step varies widely for all three strains and is greater than 100%for all ten CTM lots. This appears to be due to a systematic downwardbias of titers for Centrifuge Peak Fraction Pools (as suggested bythe >100 yield of the next step), thought to be related to the highsucrose levels in these samples.

Comments on CTM-1 Lots

In the various embodiments and example herein it is preferable that noyolk contamination take place of the harvest fluids which could occur,e.g., with improper harvest machine settings.

0.2 micron filtration step: The batch record in effect specified a PallKleenpak disposable filter, however this was not used for any CTM lots.Lots CAZ025-030 used a cartridge filter (AB1DFL7PH4-Pall hydrophilicPVDF Fluorodyne II filter) with housing instead of the Kleenpak. Thefilters had similar materials of construction (hydrophilic PVDF),however the filter areas were 5100 cm² for the Fluorodyne II cartridgefilter vs. 1500 cm² for the Kleenpak Fluorodyne II filter. For lotsCAZ031/032/033/034 a disposable Pall NovaSip C3DFLP1 filter assembly wasused instead of the Kleenpak. The membrane surface area for the C3DFLP1filter was the same as the Kleenpak filter (1500 cm²).

Temperature excursions: During the second incubation step for CAZ031,the temperature rose to 34.4° C. for 13 hours and for CAZ032 thetemperature dropped to 31.5° C. for 4 hours. These lots reached normalharvest titers above 9.0 log10 TCID50/mL, so the excursions were notregarded as having impacted the product.

Summary

Based on two Clinical Trial Manufacturing Campaigns (CTM-1 and CTM-2),the liquid FluMist process development and manufacturing work indicatesthat the process is suitable to produce clinical supplies that passrelease specifications. Aggregate data from both CTM runs and variousdevelopment studies leads to the following estimates of median processyield:

-   -   Clarification: 50% yield    -   Ultracentrifuge purification: 50% yield    -   Peak dilution and sterile filtration: 20 to 50% yield

EXAMPLE 2 Stability Testing

A trivalent vaccine formulation was prepared comprising three differentreassortant influenza viruses (7.0+/−0.5 log₁₀ FFU/dose [approximately7.0+/−0.5 log ₁₀ TCID₅₀/dose]) and comprising 200 mM sucrose; 1% (w/v)porcine gelatin hydrolysate; 1.21% (w/v) arginine monohydrocloride[equivalent to 1% (w/v) arginine base]; and 5 mM monosodium glutamate in100 mM potassium phosphate buffer (pH 7.2). The stability of theformulation was stored at −25.0 degrees C. +/−5.0 degrees for greaterthan or equal to 24 hours, but less than or equal to two weeks and thenstored at 2-8 degrees C. for various time periods. This formulation(e.g., lot 0141500003) was determined to be stable for at least 12 weeksat 4-8 degree C. In particular, the potency for each strain of virusremained within 0.5 log ₁₀ of the beginning potency prior to 4-8 degreeC. storage.

Other studies have shown that equivalent formulations (comprising threedifferent reassortant influenza viruses (7.0+/−0.5 log₁₀ FFU/dose[approximately 7.0+/−0.5 log₁₀ TCID₅₀/dose]) and comprising 200 mMsucrose; 1% (w/v) porcine gelatin hydrolysate; 1.21% (w/v) argininemonohydrocloride [equivalent to 1% (w/v) arginine base]; and 5 mMmonosodium glutamate in 100 mM potassium phosphate buffer, pH 7.2) ofdifferent clinical lots (e.g., “Campaign 3”) are stable for about 12-15months at 5.0 (+/−3.0) degrees C.

Subsequent studies have shown that formulations comprising only 200 mMsucrose; 1% (w/v) gelatin hydrolysate; 1.21% (w/v) argininemonohydrocloride [equivalent to 1% (w/v) arginine base] in 100 mMpotassium phosphate buffer, pH 7.2, but without glutamate, hadequivalent stability as the above formulations with glutamate.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes.

What is claimed is:
 1. A refrigerator-stable live cold-adapted influenzavirus composition which comprises at least one strain of influenza virusand further comprises a stabilizer consisting of: (a) 2% to 8% sucroseweight/volume (w/v), 0.75% to 2% arginine w/v, 0.02% to 0.15% glutamatew/v, 0.05% to 2% gelatin hydrolysate, and a buffer; or (b) 2% to 8%sucrose w/v, 0.75% to 2% arginine w/v, 0.05% to 2% gelatin hydrolysate,and a buffer; wherein each amount of each of the components of thestabilizer is the final amount in the virus composition, and which viruscomposition exhibits a potency loss of less than 1.0 log over a 12 monthperiod when stored at 4° C. to 8° C.
 2. The refrigerator-stable livecold-adapted influenza virus composition of claim 1, wherein at leastone influenza virus strain is selected from: an attenuated cold-adaptedinfluenza virus, a temperature-sensitive cold-adapted influenza virus,and an attenuated cold-adapted temperature-sensitive influenza virus. 3.The refrigerator-stable live cold-adapted influenza virus composition ofclaim 1, wherein at least one influenza virus strain comprises a geneticbackbone of ca A/Ann Arbor/6/60 or ca B/Ann Arbor/1/66.
 4. Therefrigerator-stable live cold-adapted influenza virus composition ofclaim 1, wherein the stabilizer consists of 6.84% sucrose w/v, 1.21%arginine w/v, 0.094% glutamate w/v, 1% gelatin hydrolysate, and abuffer.
 5. The refrigerator-stable live cold-adapted influenza viruscomposition of claim 1, wherein the stabilizer consists of 6.84% sucrosew/v, 1.21 arginine w/v, 1% gelatin hydrolysate, and a buffer.
 6. Therefrigerator-stable live cold-adapted influenza virus composition ofclaim 1, wherein the buffer is a potassium phosphate buffer.
 7. Therefrigerator-stable live cold-adapted influenza virus composition ofclaim 4, wherein the buffer is a potassium phosphate buffer.
 8. Therefrigerator-stable live cold-adapted influenza virus composition ofclaim 6, wherein the potassium phosphate buffer comprises 1.13% (w/v)dibasic potassium phosphate and 0.48% (w/v) monobasic potassiumphosphate.
 9. The refrigerator-stable live cold-adapted influenza viruscomposition of claim 7, wherein the potassium phosphate buffer comprises1.13% (w/v) dibasic potassium phosphate and 0.48% (w/v) monobasicpotassium phosphate.
 10. The refrigerator-stable live cold-adaptedinfluenza virus composition of claim 1, wherein the arginine is argininemonohydrochloride and the glutamate is glutamic acid monosodiummonohydrate.
 11. The refrigerator-stable live cold-adapted influenzavirus composition of claim 4, wherein the arginine is argininemonohydrochloride and the glutamate is glutamic acid monosodiummonohydrate.
 12. The refrigerator-stable live cold-adapted influenzavirus composition of claim 1, wherein a vaccine formulation of thecomposition comprises between 6.5 to 7.5 log₁₀ fluorescent focus units(FFU) of each influenza virus strain per 0.2 mL dose.
 13. Therefrigerator-stable live cold-adapted influenza virus composition ofclaim 4, wherein a vaccine formulation of the composition comprisesbetween 6.5 to 7.5 log₁₀ fluorescent focus units (FFU) of each influenzavirus strain per 0.2 mL dose.
 14. The refrigerator-stable livecold-adapted influenza virus composition of claim 1, wherein thecomposition comprises at least two influenza virus strains.
 15. Therefrigerator-stable live cold-adapted influenza virus composition ofclaim 14, wherein the composition comprises three influenza virusstrains.
 16. An immunogenic composition comprising therefrigerator-stable live cold-adapted influenza virus composition ofclaim
 1. 17. A vaccine comprising the immunogenic composition of claim16.
 18. A refrigerator-stable live cold-adapted influenza virus vaccineformulation which comprises: a) at least two strains of influenza virus,wherein: i) at least one influenza virus strain comprises a geneticbackbone of ca A/Ann Arbor/6/60 and at least one influenza virus straincomprises a genetic backbone of ca B/Ann Arbor/1/66, and ii) the vaccineformulation comprises between 6.5 to 7.5 log₁₀ fluorescent focus units(FFU) of each influenza virus strain per 0.2 mL dose; and b) astabilizer consisting of 6.84% sucrose w/v, 1.21% argininemonohydrochloride w/v, 0.094% glutamic acid monosodium monohydrate w/v,1% gelatin hydrolysate, and a potassium phosphate buffer, which buffercomprises 1.13% (w/v) dibasic potassium phosphate and 0.48% (w/v)monobasic potassium phosphate; wherein each amount of each of thecomponents of the stabilizer is the final amount in the vaccineformulation, and which vaccine formulation exhibits a potency loss ofless than 1.0 log over a 12 month period when stored at 4° C. to 8° C.19. The vaccine formulation of claim 18, wherein the formulationcomprises three influenza virus strains.
 20. A kit comprising thevaccine formulation of claim 18 and instructions.
 21. A kit comprisingthe vaccine formulation of claim 19 and instructions.